Reagents and methods for detecting protein crotonylation

ABSTRACT

The invention provides an isolated peptide comprising a crotonylation site, a Kcr-specific affinity reagent that specifically binds to the peptide, and a method for detecting protein crotonylation in a sample using the reagent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/728,024 filed on Dec. 27, 2012, which claims the benefit of U.S.Provisional Application No. 61/580,468, filed on Dec. 27, 2011, and is acontinuation-in-part of U.S. application Ser. No. 13/117,154, filed May27, 2011, which claims priority to U.S. Provisional Application No.61/349,185, filed May 27, 2010, and the contents of each of which areincorporated by reference herein, in their entireties and for allpurposes.

FIELD OF THE INVENTION

This invention relates to reagents and methods for detecting proteincrotonylation. More particularly, it relates to peptides comprising acrotonylation site, and their uses to develop reagents and methodsuseful for detecting crotonylation in proteins.

BACKGROUND OF THE INVENTION

Molecular anatomy of post-translational modifications that regulatecellular processes and disease progression stands as one of the majorgoals of post-genomic biological research. To date, more than 300post-translational modifications have been described, which provide anefficient way to diversify a protein's primary structure and possiblyits functions. The remarkable complexity of these molecular networks isexemplified by modifications at the side chain of lysine, one of thefifteen ribosomally-coded amino acid residues known to be modified. Theelectron-rich and nucleophilic nature of the lysine side chain makes itsuitable for undergoing covalent post-translational modificationreactions with diverse substrates that are electrophilic. The residuecan be potentially modulated by several post-translational modificationsincluding methylation, acetylation, biotinylation, ubiquitination, andsumoylation, which have pivotal roles in cell physiology and pathology.

Histones, for example, are known to be modified by an array ofpost-translational modifications, including methylation, acetylation,ubiquitination, small ubiquitin-like modification, and ribosylation. Acombinatorial array of post-translational modifications in histones,termed the “histone code”, dictates the proteins' functions in geneexpression and chromatin dynamics. Post-translational modifications ofhistones have been studied by both biochemistry (Jenuwein, et al. 2001)and mass spectrometry (Garcia, et al. 2007; Boyne, et al. 2006;Medzihradszky, et al. 2004).

Histone is acetylated at lysine residues. Lysine acetylation is anabundant, reversible, and highly regulated post-translationalmodification. It is generally highly correlated with gene activity.While initially discovered in histones, the modification was lateridentified in non-histone proteins, such as p53. A recent proteomicsscreening showed that acetyllysine is abundant and present in substratesthat are affiliated with multiple organelles and have diverse functions.Interestingly, the modification is enriched in mitochondrial proteinsand metabolic enzymes, implying its roles in fine-tuning the organelle'sfunctions and energy metabolism. The modification plays an importantrole in diverse cellular processes, such as apoptosis, metabolism,transcription, and stress response. In addition to their roles infundamental biology, lysine acetylation and its regulatory enzymes(acetyltransferases and deacetylases) are intimately linked to aging andseveral major diseases such as cancer, neurodegenerative disorders, andcardiovascular diseases.

There remains a need for developing reagents and methods useful fordetecting post-translational modifications of histones or nonhistoneproteins linked to various diseases and disorders.

SUMMARY OF THE INVENTION

The present invention relates to the use of peptides comprising acrotonylation site to develop reagents and methods for detecting proteincrotonylation, especially site specific crotonylation.

An isolated peptide comprising a crotonylation site is provided. Thepeptide may be derived from a histone protein or a fragment thereof. Thepeptide may comprise a sequence selected from SEQ ID NOs: 11-43.

The peptide may be crotonylated at a lysine site, which is also calledcrotonyllysine residue. Examples of the crotonylated peptides includeQLATKcrAA, CQLATKcrAA, YQKcrST, CYQKcrSTELL, LLPKKcrTESHHKAK,CLLPKKcrTESHHKAKG, APAPKcrKGS, APAPKcrKGSC, APAPKKcrGS, CAPAPKKcrGS,GSKKcrA, GSKKcrAVTC, TKcrAQKKDG, AVTKcrAQKKDGC, ARTKcrQTAR, ARTKcrQTARC,APRKcrQLA, APRKcrQLATC, QLATKcrAARK, QLATKcrAARKC, AARKcrSAP,AARKcrSAPATGGC, CRLLRKcrGNYAER, CLLPKcrKTESHHKAKG, CLLPKKTESHHKcrAKG,CAVTKAQKcrKDG, CARTKQTARKcrSTG, CSGRGKcrGG and CGLGKGGAKcrRHR.

The crotonylation site may be selected from the group consisting ofhuman H1.2K33, H1.2K63, H1.2K84, H1.2K89, H1.2K96, H1.2K158, H1.2K167,H2AK36, H2AK118, H2AK119, H2AK125, H2BK5, H2BK11, H2BK12, H2BK15,H2BK16, H2BK20, H2BK23, H2BK34, H3K4, H3K9, H3K18, H3K23, H3K27, H3K56,H4K5, H4K8, H4K12 and H4K16. Preferably, the crotonylation site is humanH2AK119, H2BK11, H2BK12, H2BK16, H2BK20, H2BK34, H3K4, H3K18, H3K23, orH3K27.

A method for producing a Kcr-specific affinity reagent is also provided.The Kcr-specific affinity reagent binds specifically to a protein or afragment thereof comprising a crotonylation site.

Where the Kcr-specific affinity reagent is an antibody, the Kcr-specificaffinity reagent may be produced by immunizing a host with a peptide ofthe present invention. The peptide may comprise a sequence selected fromSEQ ID NOs: 11-43.

Where the Kcr-specific affinity reagent is a polypeptide, theKcr-specific affinity reagent may be produced by screening syntheticpeptide library using a peptide of the present invention. The syntheticpeptide library may be a phage display library or a yeast displaylibrary. The peptide may comprise a sequence selected from SEQ ID NOs:11-43.

An isolated Kcr-specific affinity reagent is further provided. Theisolated Kcr-specific affinity reagent is capable of bindingspecifically to the peptide of the present invention. The peptide maycomprise a sequence selected from SEQ ID NOs: 11-43.

The binding of the Kcr-specific affinity reagent may be dependent on thecrotonylation site, but not its surrounding peptide sequence. It mayrecognize a polypeptide comprising a crotonyllysine residue.

The binding of the Kcr-specific affinity reagent may be dependent on thecrotonylation site and its surrounding peptide sequence. It may bindspecifically to the peptide when crotonylated at the crotonylation site,but not the peptide when not crotonylated at the crotonylation site. Thecrotonylation site may be selected from the group consisting of humanH1.2K33, H1.2K63, H1.2K84, H1.2K89, H1.2K96, H1.2K158, H1.2K167, H2AK36,H2AK118, H2AK119, H2AK125, H2BK5, H2BK11, H2BK12, H2BK15, H2BK16,H2BK20, H2BK23, H2BK34, H3K4, H3K9, H3K18, H3K23, H3K27, H3K56, H4K5,H4K8, H4K12 and H4K16, preferably H2AK119, H2BK11, H2BK12, H2BK16,H2BK20, H2BK34, H3K4, H3K18, H3K23, and H3K27.

A method for detecting protein crotonylation in a sample is furtherprovided. The detection method comprises contacting the sample with anisolated Kcr-specific affinity reagent of the present invention to forma binding complex. The Kcr-specific affinity reagent binds specificallyto a protein or a fragment thereof comprising a crotonylation site whenthe protein or a fragment thereof is crotonylated at the crotonylationsite. The method further comprises detecting the binding complex. Thepresence of the binding complex indicates protein crotonylation in thesample.

A kit for detecting protein crotonylation in a sample is furtherprovided. The kit comprises an isolated Kcr-specific affinity reagent ofthe present invention. The Kcr-specific affinity reagent bindsspecifically to a protein or a fragment thereof comprising acrotonylation site when the protein or a fragment thereof iscrotonylated at the crotonylation site.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 summarizes an experimental strategy and results for identifyinghistone PTM sites according to some embodiments of the presentinvention. (A) A schematic diagram illustrating the experimental designfor comprehensive mapping of PTM sites in linker and core histones fromHeLa cells using Methods I, II, III or IV. (B) Peptide sequence coverageof linker and core histones in Methods I, II, III or IV. (C) A summaryof the identified PTM sites. Abbreviations: Kme, lysine monomethylation;Kme2, lysine dimethylation; Kfo, lysine formylation; Kac, lysineacetylation; Rme, arginine monomethylation; Yoh, tyrosine hydroxylation;and Kcr, lysine crotonylation. (D) A diagram showing the identifiedsites of histone PTMs other than lysine crotonylation (Kcr). Amino acidresidue number is indicated below its sequence. Gray and blank boxesindicate N-terminal and globular core domains, respectively. (E)Illustrations of histone Kcr sites in human HeLa cells and mouse MEFcells. All newly discovered Kcr sites are shown underlined. Previouslyreported lysine acetylation (Kac) sites are also shown.

FIG. 2 shows short-chain lysine acylations resulting in a crotonyllysineand a acetyllysine. (A) An illustration of the enzymatic reactions forlysine acetylation by lysine acetyltransferases (KATs) using acetyl-CoAas a cofactor, and a hypothesized mechanism for Kcr using crotonyl-CoAas a cofactor. (B) Ball-and-stick models of a crotonyl group and anacetyl group. The three-dimensional arrangement of four carbons and oneoxygen of the crotonyl group are rigid and located in the same plane(left panel). The two olefinic carbons of the crotonyl group are shownin yellow. In contrast, the tetrahedral CH3 in the acetyl group (rightpanel) can be rotated such that it is structurally very different fromthe crotonyl group. (C) Chemical structure of vinylacetyllysine(but-3-enoyllysine), methacryllysine, and cyclopropanecarboxyllysine.(D) Crotonyl-CoA metabolism pathways. Crotonyl-CoA was generated frombutyryl-CoA or glutaryl-CoA, and oxidized to acetyl-CoA through multiplesteps.

FIG. 3 shows identification and verification of a Kcr peptide,PEPAKcrSAPAPK (SEQ ID NO: 44), where “Kcr” represents a crotonyllysineresidue. (A-C) High-resolution MS/MS spectrum of a tryptic peptide,PEPAKSAPAPK, with a mass of +68.0230 Da at its Lys5 residue identifiedfrom in vivo histone H2B (A), its synthetic Kcr counterpart (B), and apeptide mixture of the in vivo-derived tryptic peptide and its syntheticcounterpart (C), each showing the same MS/MS fragmentation patterns andthe same precursor ion mass. Inset shows their precursor ion masses. (D)Extracted ion chromatograms (XICs) of the in vivo-derivedPEPAK+68.0230SAPAPK peptide, the synthetic Kcr counterpart, and theirmixture by nano-HPLC/MS/MS analysis using a reversed-phase HPLC column,showing the coelution of the two peptides.

FIG. 4 shows lysine crotonylation (Kcr) in histones. (A) Dot-spot assayusing five peptide libraries, with the relative amounts as indicated,detected by an anti-Kcr pan antibody. Each peptide library contains 13residues CXXXXXKXXXXXX (SEQ ID NO: 45), where X is a mixture of 19 aminoacids (excluding cysteine), C is cysteine, and the 7th residue is afixed lysine residue: unmodified lysine (K), Kac, propionyllysine (Kpr),butyryllysine (Kbu), or Kcr. (B) BSA, vinylacetyl-K BSA, methacryl-K BSAand crotonyl-K BSA detected by an anti-Kcr pan antibody by Westernblotting (top panel) and by blue stain (bottom panel). (C) Core histoneproteins H2A, H2B, H3, H4 and linker histone H1 with completion of apeptide library bearing an unmodified lysine (K), methacryl-K or Kcr asdetected by Western blotting with an anti-Kcr pan antibody (top panel)or blue stain (bottom panel). (D) Lysine crotonylation signal in corehistone proteins H2A, H2B, H3, H4 and linker histone H1 were detected byWestern blotting with an anti-Kcr pan antibody (top panel) or blue stain(bottom panel) with competition of a peptide library bearing anunmodified lysine (K), Kac, Kpr, Kbu or Kcr.

FIG. 5 shows lysine crotonylation (Kcr) in proteins by in vivoD4-crotonate isotopic labeling. (A) Dynamics of histone Kcr in responseto crotonate. Histone proteins extracted from human prostate cancer cellline Du145 were incubated with 0, 50, or 100 mM crotonate for 24 hr, andthen western blotted with an anti-Kcr pan antibody. (B) MS/MS spectrumof PEPA KD4-crSAPAPK identified from a D4-crotonate-labeled sample. Amixture of D4-, D3- and D2-crotonyl groups was used for theidentification of D4-crotonyl peptide.

FIG. 6 shows histone crotonylation in different cell types. Kcr signalsin core histones of H. sapiens (HeLa), M. musculus (MEF), C. elegans, D.melanogaster (S2), and S. cerevisiae cells by western blotting analysiswith competition of lysine (K) (left panel) or crotonyllysine (Kcr)(right panel).

FIG. 7 shows crotonylation in HeLa cell lysates detected by Westernblotting with an anti-Kcr pan antibody (left panel) and blue staining(right panel).

FIG. 8 shows crotonylation in Hela histones detected by Western blottingwith sequence-specific antibodies against H3K56 (anti-H3K56cr; toppanel) or H3K23 (anti-H3K23cr; middle panel), and by blue stain (bottompanel).

FIG. 9 shows protein sequences of human histone proteins (A) H1.2 (SEQID NO: 1), (B) H2A (SEQ ID NO: 2), (C) H2B (SEQ ID NO: 3), (D) H3 (SEQID NO: 4), and (E) H4 (SEQ ID NO: 5).

FIG. 10 shows protein sequences of mouse histone proteins (A) H1.2 (SEQID NO: 6), (B) H2A (SEQ ID NO: 7), (C) H2B (SEQ ID NO: 8), (D) H3 (SEQID NO: 9), and (E) H4 (SEQ ID NO: 10).

FIG. 11 shows detection of (A) crotonylated H2A peptide at Lys119 (lane1), crotonylated H2A peptide at Lys 18 (lane 2) and unmodified H2Apeptide at Lys119 (lane 3) using anti-crotonyl-histone H2A (Lys 119)rabbit pAb in dot blotting analysis, and (B) 30 μg of crude proteinsfrom HeLa whole cell lysates without (left) or with (right) treatment ofsodium crotonylate treatment (30 mM, 4 hours) usinganti-crotonyl-histone H2A (Lys 119) rabbit pAb (1:2000) in Westernblotting analysis.

FIG. 12 shows detection of (A) crotonylated H2B peptide at Lys11 (lane1), crotonylated H2B peptide at Lys12 (lane 2) and unmodified H2Bpeptide at Lys11 (lane 3) using anti-crotonyl-histone H2B (Lys11) rabbitpAb in dot blotting analysis, and (B) 30 μg of crude proteins from HeLawhole cell lysates without (left) or with (right) treatment of sodiumcrotonylate treatment (30 mM, 4 hours) using anti-crotonyl-histone H2B(Lys11) rabbit pAb (1:2000) in Western blotting analysis.

FIG. 13 shows detection of (A) crotonylated H2B peptide at Lys 12 (lane1), crotonylated H2B peptide at Lys12 (lane 2) and unmodified H2Bpeptide at Lys12 (lane 3) using anti-crotonyl-histone H2B (Lys112)rabbit pAb in dot blotting analysis, and (B) 30 μg of crude proteinsfrom HeLa whole cell lysates without (left) or with (right) treatment ofsodium crotonylate treatment (10 mM, 4 hours) usinganti-crotonyl-histone H2B (Lys12) rabbit pAb (1:1000) in Westernblotting analysis.

FIG. 14 shows detection of (A) crotonylated H2B peptide at Lys16(lane 1) and unmodified H2B peptide at Lys16 (lane 2) usinganti-crotonyl-histone H2B (Lys16) rabbit pAb in dot blotting analysis,and (B) 30 μg of crude proteins from HeLa whole cell lysates without(left) or with (right) treatment of sodium crotonylate treatment (30 mM,4 hours) using anti-crotonyl-histone H2B (Lys16) rabbit pAb (1:1000) inWestern blotting analysis.

FIG. 15 shows detection of (A) crotonylated H2B peptide at Lys20 (lane1), butyrylated H2B peptide at Lys20 (lane 2), crotonylated H2B at Lys23(lane 3) and unmodified H2B peptide at Lys12 (lane 4) usinganti-crotonyl-histone H2B (Lys20) rabbit pAb in dot blotting analysis,and (B) 30 μg of crude proteins from HeLa whole cell lysates without(left) or with (right) treatment of sodium crotonylate treatment (10 mM,4 hours) using anti-crotonyl-histone H2B (Lys20) rabbit pAb (1:2000) inWestern blotting analysis.

FIG. 16 shows detection of (A) crotonylated H2B peptide at Lys34 (lane1), succinylated H2B peptide at Lys34 (lane 2) and unmodified H2Bpeptide at Lys34 (lane 3) using anti-crotonyl-histone H2B (Lys34) rabbitpAb in dot blotting analysis, and (B) 30 μg of crude proteins from HeLawhole cell lysates without (left) or with (right) treatment of sodiumcrotonylate treatment (30 mM, 4 hours) using anti-crotonyl-histone H2B(Lys34) rabbit pAb (1:2000) in Western blotting analysis.

FIG. 17 shows detection of (A) crotonylated H3 peptide at Lys4 (lane 1),crotonylated H3 peptide at Lys9 (lane 2), butyrylated H3 peptide at Lys9(lane 3), and unmodified H3 peptide at Lys4 (lane 4) usinganti-crotonyl-histone H3 (Lys4) rabbit pAb in dot blotting analysis, and(B) 30 μg of crude proteins from HeLa whole cell lysates without (left)or with (right) treatment of sodium crotonylate treatment (30 mM, 4hours) using anti-crotonyl-histone H3 (Lys4) rabbit pAb (1:2000) inWestern blotting analysis.

FIG. 18 shows detection of (A) crotonylated H3 peptide at Lys4 (lane 1),butyrylated H3 peptide at Lys9 (lane 2), crotonylated H3 peptide at Lys9(lane 3) and unmodified H3 peptide at Lys4 (lane 4) usinganti-crotonyl-histone H3 (Lys4) mouse mAb in dot blotting analysis, and(B) 30 g of crude proteins from HeLa whole cell lysates without (left)or with (right) treatment of sodium crotonylate treatment (30 mM, 4hours) using anti-crotonyl-histone H3 (Lys4) mouse mAb (1:2000) inWestern blotting analysis.

FIG. 19 shows detection of (A) crotonylated H3 peptide at Lys18 (lane1), butyrylated H3 peptide at Lys18 (lane 2), propionated H3 peptide atLys18 (lane 3), and unmodified H3 peptide at Lys18 (lane 4) usingcrotonyl-histone H3 (Lys18) rabbit pAb in dot blotting analysis, and (B)30 μg of crude proteins from HeLa whole cell lysates without (left) orwith (right) treatment of sodium crotonylate treatment (30 mM, 4 hours)using anti-crotonyl-histone H3 (Lys18) rabbit pAb (1:1000) in Westernblotting analysis.

FIG. 20 shows detection of (A) crotonylated H3 peptide at Lys23 (lane1), butyrylated H3 peptide at Lys23 (lane 2), propionated H3 peptide atLys23 (lane 3), and unmodified H3 peptide at Lys23 (lane 4) usingcrotonyl-histone H3 (Lys23) mouse mAb in dot blotting analysis, and (B)30 μg of crude proteins from HeLa whole cell lysates without (left) orwith (right) treatment of sodium crotonylate treatment (30 mM, 4 hours)using anti-crotonyl-histone H3 (Lys23) mouse mAb (1:1000) in Westernblotting analysis.

FIG. 21 shows detection of (A) crotonylated H3 peptide at Lys23 (lane1), butyrylated H3 peptide at Lys23 (lane 2), propionated H3 peptide atLys23 (lane 3), and unmodified H3 peptide at Lys23 (lane 4) usingcrotonyl-histone H3 (Lys23) rabbit pAb in dot blotting analysis, and (B)30 μg of crude proteins from HeLa whole cell lysates without (left) orwith (right) treatment of sodium crotonylate treatment (30 mM, 4 hours)using anti-crotonyl-histone H3 (Lys23) rabbit pAb (1:1000) in Westernblotting analysis.

FIG. 22 shows detection of (A) crotonylated H3 peptide at Lys27 (lane1), acetylated H3 peptide at Lys27 (lane 2), and unmodified H3 peptideat Lys27 (lane 3) using crotonyl-histone H3 (Lys27) mouse mAb in dotblotting analysis, and (B) 30 g of crude proteins from HeLa whole celllysates without (left) or with (right) treatment of sodium crotonylatetreatment (30 mM, 4 hours) using anti-crotonyl-histone H3 (Lys27) mousemAb (1:2000) in Western blotting analysis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of novel lysinecrotonylation sites in proteins. In particular, peptides derived fromhistone proteins or fragments thereof comprising a crotonylation siteare used to generate reagents useful for detecting proteincrotonylation, especially for detecting site specific proteincrotonylation.

The term “peptide” used herein refers to a linear chain of two or moreamino acids linked by peptide bonds. A peptide may have about 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40,50, 100, 200 or more amino acids. The amino acids of a peptide may bemodified, deleted, added or substituted. A peptide may be obtained usingconventional techniques known in the art. For example, a peptide may besynthesized or obtained from a native or recombinant protein byenzymatic digestion.

The term “polypeptide” used herein refers to a peptide having at least 4amino acids, preferably at least about 20 amino acids, regardless ofpost-translational modification. The term “protein” used herein refersto a biological molecule consisting of one or more polypeptides,regardless of post-translational modification. Each polypeptide in aprotein may be a subunit. The polypeptide or protein may be in a nativeor modified form, and may exhibit a biological function orcharacteristics.

Where a protein is a single polypeptide, the terms “protein” and“polypeptide” are used herein interchangeably. A fragment of apolypeptide or protein refers to a portion of the polypeptide or proteinhaving an amino acid sequence that is the same as a part, but not all,of the amino acid sequence of the polypeptide or protein. Preferably, afragment of a polypeptide or protein exhibits a biological function orcharacteristics identical or similar to that of the polypeptide orprotein.

The term “derived from” used herein refers to the origin or source fromwhich a biological molecule is obtained, and may include naturallyoccurring, recombinant, unpurified or purified molecules. A biologicalmolecule such as a peptide (e.g., a polypeptide or protein) may bederived from an original molecule, becoming identical to the originalmolecule or a variant of the original molecule. For example, a peptidederived from an original peptide may have an amino acid sequenceidentical or similar to the amino acid sequence of its original peptide,with at least one amino acid modified, deleted, inserted, orsubstituted. A derived peptide may have an amino acid sequence at leastabout 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%,preferably at least about 50%, more preferably at least about 80%, mostpreferably at least about 90%, identical to the amino acid sequence ofits original peptide, regardless of post-translational modification.Preferably, a derived biological molecule (e.g., a peptide) may exhibita biological function or characteristics identical or similar to that ofthe original biological molecule.

The term “Kcr-specific affinity reagent” used herein refers to amolecule that is capable of binding to a protein having one or morecrotonyllysine residues but not its unmodified counterpart. TheKcr-specific affinity reagent may be a peptide, polypeptide or protein.For example, the Kcr-specific affinity reagent may be an antibody.

The term “antibody” used herein includes whole antibodies, and antigenbinding fragments (or antigen-binding portions) and single chainsthereof. A whole antibody refers to a glycoprotein typically having twoheavy chains and two light chains, and includes an antigen bindingportion. The term “antigen binding portion” of an antibody used hereinrefers to one or more fragments of the antibody that retain the abilityof specifically binding to an antigen. The term “single-chain variablefragment” of an antibody used herein refers to a fusion protein of thevariable regions of the heavy and light chains of the antibody,connected with a short linker peptide, for example, of about 20-25 aminoacids, that retains the ability of specifically binding to an antigen.

An isolated peptide comprising a crotonylation site is provided. Thepeptide may be derived from a protein, for example, a histone protein,or a fragment thereof comprising a crotonylation site. The peptide maybe crotonylated or not crotonylated at the crotonylation site.

The peptide of the present invention may have at least about 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70,80, 90, 100, 150 or 200 amino acids. The peptide may have about 3-25amino acids, preferably 5-20 amino acids, more preferably 6-14 aminoacids.

The term “crotonylation” used herein refers to substitution of ahydrogen atom in a molecule (e.g., lysine) with a crotonyl group. Forexample, lysine may be crotonylated and become crotonyllysine.

A histone protein may be obtained from a eukaryotic cell. Examples of aeukaryotic cell include cells from a yeast (e.g., S. cerevisiae), an C.elegans, a Drosophila (e.g., D. melanogaster (S2)), a mouse (e.g., M.musculus (MEF)), or a human. Preferably, the eukaryotic cell is amammalian cell, for example, a human, primate, mouse, rat, horse, cow,pig, sheep, goat, chicken, dog or cat cell. More preferably, theeukaryotic cell is a human cell.

A histone protein may be a histone linker protein or a histone coreprotein. A histone linker protein may be selected from the members ofthe H1 family, including the H1F subfamily (e.g., H1F0, H1FNT, HIFOO,and H1FX) and the H1H1 subfamily (e.g., HIST1H1A, HIST1H1B, HIST1H1C,HIST1H1D, HIST1H1E and HIST1H1T). A histone core protein may a member ofthe H2A, H2B, H3 or H4 family. A histone core protein in the H2A familymay be a member of the H2AF subfamily (e.g., H2AFB1, H2AFB2, H2AFB3,H2AFJ, H2AFV, H2AFX, H2AFY, H2AFY2, and H2AFZ), the H2A1 subfamily(e.g., HIST1H2AA, HIST1H2AB, HIST1H2AC, HIST1H2AD, HIST1H2AE, HIST1H2AG,HIST1H2AI, HIST1H2AJ, HIST1H2AK, HIST1H2AL, and HIST1H2AM), or the H2A2subfamily (e.g., HIST2H2AA3, and HIST2H2AC). A histone core protein inthe H2B family may be a member of the H2BF subfamily (e.g., H2BFM,H2BFO, H2BFS, and H2BFWT), the H2B1 subfamily (e.g., HIST1H2BA,HIST1H2BB, HIST1H2BC, HIST1H2BD, HIST1H2BE, HIST1H2BF, HIST1H2BG,HIST1H2BH, HIST1H2BI, HIST1H2BJ, HIST1H2BK, HIST1H2BL, HIST1H2BM,HIST1H2BN, and HIST1H2BO), or the H2B2 subfamily (e.g., HIST2H2BE). Ahistone core protein in the H3 family may be a member of the H3A1subfamily (e.g., HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E,HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, and HIST1H3J), the H3A2subfamily (e.g., HIST2H3C), or the H3A3 subfamily (e.g., HIST3H3). Ahistone core protein in the H4 family may be a member of the H41subfamily (e.g., HIST1H4A, HIST1H4B, HIST1H4C, HIST1H4D, HIST1H4E,HIST1H4F, HIST1H4G, HIST1H4H, HIST1H4I, HIST1H4J, HIST1H4K, andHIST1H4L), or the H44 subfamily (e.g., HIST4H4).

The protein and gene sequences of histone proteins in various speciesare known in the art. The protein sequences of human H1.2, H2A, H2B, H3and H4 histone proteins can be found in GenBank database Accession Nos.P16403 (H12_HUMAN) having SEQ ID NO: 1 (FIG. 9A), P04908 (H2A1B_HUMAN)having SEQ ID NO: 2 (FIG. 9B), P33778 (H2B1B_HUMAN) having SEQ ID NO: 3(FIG. 9C), P84243 (H33_HUMAN) having SEQ ID NO: 4 (FIG. 9D) and P62805(H4_HUMAN) having SEQ ID NO: 5 (FIG. 9E) respectively. The full-lengthprotein sequences of mouse histone proteins H1.2, H2A, H2B, H3 and H4can be found in the GenBank database Accession Nos. P15864 (H12_MOUSE)having SEQ ID NO: 6 (FIG. 10A), P22752 (H2A1_MOUSE) having SEQ ID NO: 7(FIG. 10B), Q64475 (H2B1B_MOUSE) having SEQ ID NO: 8 (FIG. 10C), P84244(H33_MOUSE) having SEQ ID NO: 9 (FIG. 10D) and P62806 (H4_MOUSE) havingSEQ ID NO: 10 (FIG. 10E), respectively.

A histone protein may be crotonylated at a crotonylation site. Acrotonylation site may be lysine 33 (H1.2K33), lysine 63 (H1.2K63),lysine 84 (H1.2K84), lysine 89 (H1.2K89), lysine 96 (H1.2K96), lysine158 (H1.2K158), or lysine 167 (H1.2K167) in a human H1.2 histone protein(SEQ ID NO: 1); lysine 36 (H2AK36), lysine 118 (H2AK118), lysine 119(H2AK119), or lysine 125 (H2AK125) in human H2A histone protein (SEQ IDNO: 2); lysine 5 (H2BK5), lysine 11 (H2BK 1), lysine 12 (H2BK12), lysine15 (H2BK15), lysine 16 (H2BK16), lysine 20 (H2BK20), lysine 23 (H2BK23),or lysine 34 (H2BK34) in human H2B histone protein (SEQ ID NO: 3);lysine 4 (H3K4), lysine 9 (H3K9), lysine 18 (H3K18), lysine 23 (H3K23),lysine 27 (H3K27), or lysine 56 (H3K56) in human H3 histone protein (SEQID NO: 4); or lysine 5 (H4K5), lysine 8 (H4K8), lysine 12 (H4K12), orlysine 16 (H4K16) in human H4 histone protein (SEQ ID NO: 5).

Many crotonylation sites in histone proteins are conserved amongdifferent species (FIG. 1E). A mouse H1.2 histone protein (SEQ ID NO: 6)comprises crotonylation sites at lysine 33 (H1.2K33), lysine 63(H1.2K63), lysine 84 (H1.2K84), lysine 158 (H1.2K158), and lysine 167(H1.2K167) of SEQ ID NO: 6, corresponding to human H1.2K33, H1.2K63,H1.2K84, H1.2K158 and H1.2K167, respectively. A mouse H2A histoneprotein (SEQ ID NO: 7) comprises crotonylation sites at lysine 36(H2AK36), and lysine 118 (H2AK118) of SEQ ID NO: 7, corresponding tohuman H2AK36 and H2AK118, respectively. A mouse H2B histone protein (SEQID NO: 8) comprises crotonylation sites at lysine 5 (H2BK5), lysine 11(H2BK11), lysine 12 (H2BK12), lysine 15 (H2BK15), lysine 16 (H2BK16),lysine 20 (H2BK20), lysine 23 (H2BK23), and lysine 34 (H2BK34) of SEQ IDNO: 8, corresponding to human H2BK5, H2BK 11, H2BK12, H2BK15, H2BK16,H2BK20, H2BK23, and H2BK34, respectively. A mouse H3 histone protein(SEQ ID NO: 9) comprises a crotonylation site comprises lysine 4 (H3K4),lysine 9 (H3K9), lysine 18 (H3K18), lysine 23 (H3K23), lysine 27(H3K27), and lysine 56 (H3K56) of SEQ ID NO: 9, corresponding to humanH3K4, H3K9, H3K18, H3K23, H3K27, H3K56, respectively. A mouse H4 histoneprotein crotonylation site (SEQ ID NO: 10) comprises crotonylation sitesat lysine 5 (H4K5), lysine 8 (H4K8), and lysine 16 (H4K16) of SEQ ID NO:10, of which mouse H4K5 and H4K8 correspond to the human H4K5 and H4K8,respectively.

A fragment of a histone protein may have an amino acid sequence that isthe same as a part, not all, of the amino acid sequence of the histoneprotein comprising at least one crotonylation site in the histoneprotein. A histone protein fragment may have at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50,60, 70, 80, 90, 100, 150 or 200 amino acids. A fragment of a histoneprotein may have about 3-25 contiguous amino acids, preferably about5-20 contiguous amino acids, more preferably about 6-14 contiguous aminoacids, of the histone protein covering at least one crotonylation sitein the histone protein.

An isolated peptide of the present invention may be prepared usingconventional techniques known in the art. The peptide may be a histonepeptide derived from a histone protein or a fragment thereof comprisinga crotonylation site. A histone protein may be obtained from abiological sample or prepared using recombinant techniques. A fragmentof a histone protein may be prepared by recombinant techniques, or bydigesting the histone protein with an enzyme (e.g., trypsin). Thehistone protein or a fragment thereof may be crotonylated naturally orartificially at a crotonylation site. The presence of a crotonyllysinemay be confirmed by using conventional techniques known in the art, forexample, mass spectrometry.

An isolated peptide of the present invention may comprise a sequenceselected from the group consisting of SEQ ID NOs: 11-43. The peptidesmay encompass various regions and crotonylation sites in human histoneproteins (Table 1). These peptides may comprise a crotonylated lysineresidue (Kcr) (also known as a crotonyllysine residue). Examples ofcrotonylated peptides covering crotonylation sites in human histoneproteins are shown in Table 1.

A method for producing a Kcr-specific affinity reagent is provided. TheKcr-specific affinity reagent specifically binds to a protein or afragment thereof comprising a crotonyllysine. The protein may be ahistone protein.

The Kcr-specific affinity reagent may be an antibody produced bydifferent methods known in the art. For example, the production methodmay comprise immunizing a host with an antigenic peptide to produce theantibody. The method may further comprise collecting antisera from thehost. The antigenic peptide may be derived from a histone protein or afragment thereof comprising the crotonylation site. The antigenicpeptide may comprise a peptide of the present invention. The antigenicpeptide may be crotonylated or not crotonylated. Examples of thenon-crotonylated antigenic peptides include QLATKAA, CQLATKAA, YQKST,CYQKSTELL, LLPKKTESHHKAK, CLLPKKTESHHKAKG, APAPKKGS, APAPKKGSC,CAPAPKKGS, GSKKA, GSKKAVTC, TKAQKKDG, AVTKAQKKDGC, ARTKQTAR, ARTKQTARC,APRKQLA, APRKQLATC, QLATKAARK, QLATKAARKC, AARKSAP, AARKSAPATGGC,CRLLRKGNYAER, CAVTKAQKKDG, CARTKQTARKSTG, CSGRGKGG and CGLGKGGAKRHR.Examples of the crotonylated antigenic peptides include QLATKcrAA,CQLATKcrAA, YQKcrST, CYQKcrSTELL, LLPKKcrTESHHKAK, CLLPKKcrTESHHKAKG,APAPKcrKGS, APAPKcrKGSC, APAPKKcrGS, CAPAPKKcrGS, GSKKcrA, GSKKcrAVTC,TKcrAQKKDG, AVTKcrAQKKDGC, ARTKcrQTAR, ARTKcrQTARC, APRKcrQLA,APRKcrQLATC, QLATKcrAARK, QLATKcrAARKC, AARKcrSAP, AARKcrSAPATGGC,CRLLRKcrGNYAER, CLLPKcrKTESHHKAKG, CLLPKKTESHHKcrAKG, CAVTKAQKcrKDG,CARTKQTARKcrSTG, CSGRGKcrGG and CGLGKGGAKcrRHR. The N-terminal orC-terminal end of any of these peptides may be extended by 1-20residues, depending on the proteolytic enzymes used for digestion ofhistone proteins.

The method may further comprise purifying the antibody from theantisera. The method may further comprise utilizing spleen cells fromthe host to generate a monoclonal antibody. In some embodiments, theantibody specifically binds to a histone protein or a fragment thereofwhen the histone protein or a fragment thereof is crotonylated at thecrotonylation site, and does not bind to the histone protein or afragment thereof when the histone protein or a fragment thereof is notcrotonylated at the crotonylation site. In other embodiments, theantibody specifically binds to the histone protein or a fragment thereofwhen the histone protein or a fragment thereof is not crotonylated atthe crotonylation site, and does not bind to the histone protein or afragment thereof when the histone protein or a fragment thereof iscrotonylated at the crotonylation site.

The host may be a mammal suitable for producing antibodies. For example,the host may be a mouse, rabbit, or goat.

The Kcr-specific affinity reagent may be a polypeptide. The polypeptidemay be produced by screening a synthetic peptide library using a peptidecomprising the peptide of the present invention. The synthetic peptidelibrary may be a phage display library, a yeast display library or othersynthetic peptide library comprising peptides having randomized aminoacid sequences.

Also provided is an isolated Kcr-specific affinity reagent thatspecifically binds to a peptide of the present invention. TheKcr-specific affinity reagent may be an antibody or a polypeptidederived by screening a synthetic peptide library. In some embodiments,the Kcr-specific affinity reagent specifically binds to the peptide whencrotonylated, and does not bind to the peptide when not crotonylated.The binding of the Kcr-specific affinity reagent to a peptide of thepresent invention may be dependent on the crotonylation site, but notits surrounding peptide sequence. For example, the Kcr-specific affinityreagent is an anti-crotonylation pan antibody.

The binding of the Kcr-specific affinity reagent to a peptide of thepresent invention may be dependent on the crotonylation site and itssurrounding peptide sequence. It may recognize a polypeptide having acrotonyllysine residue. The recognition may depend on not only thecrotonyllysine residue but also its surrounding 1-5 residues. In someembodiments, the crotonylation site specific Kcr-specific affinityreagent specifically binds to a histone protein or a fragment thereofwhen the histone protein or a fragment thereof is crotonylated at thecrotonylation site, and does not bind to the histone protein or afragment thereof when the histone protein or a fragment thereof is notcrotonylated at the crotonylation site. Examples of crotonylation sitespecific Kcr-specific affinity reagents include anti-H3K23cr rabbit pAb,anti-H3K56cr rabbit pAb, anti-crotonyl-histone H2A (Lys119) rabbit pAb,anti-crotonyl-histone H2B (Lys11) rabbit pAb, anti-crotonyl-histone H2B(Lys12) rabbit pAb, anti-crotonyl-histone H2B (Lys16) rabbit pAb,anti-crotonyl-histone H2B (Lys20) rabbit pAb, anti-crotonyl-histone H2B(Lys34) rabbit pAb, anti-crotonyl-histone H3 (Lys4) rabbit pAb,anti-crotonyl-histone H3 (Lys4) mouse mAb, anti-crotonyl-histone H3(Lys18) rabbit pAb, anti-crotonyl-histone H3 (Lys23) mouse mAb,anti-crotonyl-histone H3 (Lys23) rabbit pAb, and anti-crotonyl-histoneH3 (Lys27) mouse mAb.

With respect to a Kcr-specific affinity reagent of the presentinvention, the crotonylation site may be any crotonylation site in anyhistone protein from any species. Examples of the crotonylation sitesinclude human crotonylation sites H1.2K33, H1.2K63, H1.2K84, H1.2K89,H1.2K96, H1.2K158, H1.2K167, H2AK36, H2AK118, H2AK119, H2AK125, H2BK5,H2BK11, H2BK12, H2BK15, H2BK16, H2BK20, H2BK23, H2BK34, H3K4, H3K9,H3K18, H3K23, H3K27, H3K56, H4K5, H4K8, H4K12 and H4K16, mousecrotonylation sites H1.2K33, H1.2K63, H1.2K84, H1.2K158, H1.2K167,H2AK36, H2AK18, H2BK5, H2BK11, H2BK12, H2BK15, H2BK16, H2BK20, H2BK23,H2BK34, H3K4, H3K9, H3K18, H3K23, H3K27, H3K56, H4K5, H4K8, and H4K16,and homologous lysine sites in corresponding eukaryotic histoneproteins.

The Kcr-specific affinity reagent may specifically bind to a human H1.2histone protein (SEQ ID NO: 1) or a fragment thereof when the human H1.2histone protein or a fragment thereof is crotonylated at a crotonylationsite selected from the group consisting of human H1.2K33, H1.2K63,H1.2K84, H1.2K89, H1.2K96, H1.2K158 and H1.2K167, but not when the H1.2histone protein or a fragment thereof is not crotonylated at thecrotonylation site.

The Kcr-specific affinity reagent may specifically bind to a human H2Ahistone protein (SEQ ID NO: 2) or a fragment thereof when the human H2Ahistone protein or a fragment thereof is crotonylated at a crotonylationsite selected from the group consisting of human H2AK36, H2AK18, H2AK119(e.g., anti-crotonyl-histone H2A (Lys 119) rabbit pAb) and H2AK125, butnot when the H2A histone protein or a fragment thereof is notcrotonylated at the crotonylation site.

The Kcr-specific affinity reagent may specifically bind to a human H2Bhistone protein (SEQ ID NO: 3) or a fragment thereof when the human H2Bhistone protein or a fragment thereof is crotonylated at a crotonylationsite selected from the group consisting of human H2BK5, H2BK11 (e.g.,anti-crotonyl-histone H2B (Lys11) rabbit pAb), H2BK12 (e.g.,anti-crotonyl-histone H2B (Lys12) rabbit pAb), H2BK15, H2BK16 (e.g.,anti-crotonyl-histone H2B (Lys16) rabbit pAb), H2BK20 (e.g.,anti-crotonyl-histone H2B (Lys20) rabbit pAb), H2BK23 and H2BK34 (e.g.,anti-crotonyl-histone H2B (Lys34) rabbit pAb), but not when the H2Bhistone protein or a fragment thereof is not crotonylated at thecrotonylation site.

The Kcr-specific affinity reagent may specifically bind to a human H3histone protein (SEQ ID NO: 4) or a fragment thereof when the human H3histone protein or a fragment thereof is crotonylated at a crotonylationsite selected from the group consisting of human H3K4 (e.g.,anti-crotonyl-histone H3 (Lys4) rabbit pAb and anti-crotonyl-histone H3(Lys4) mouse mAb), H3K9, H3K18 (e.g., anti-crotonyl-histone H3 (Lys18)rabbit pAb), H3K23 (e.g., anti-H3K23cr rabbit pAb, anti-crotonyl-histoneH3 (Lys23) rabbit pAb, and anti-crotonyl-histone H3 (Lys23) mouse mAb),H3K27 (e.g., anti-crotonyl-histone H3 (Lys27) mouse mAb) and H3K56(e.g., anti-H3K56cr rabbit pAb), but not when the H3 histone protein ora fragment thereof is not crotonylated at the crotonylation site.

The Kcr-specific affinity reagent may specifically bind to a human H4histone protein (SEQ ID NO: 5) or a fragment thereof when the human H4histone protein or a fragment thereof is crotonylated at a crotonylationsite selected from the group consisting of human H4K5, H4K8, H4K12 andH4K16, but not when the H4 histone protein or a fragment thereof is notcrotonylated at the crotonylation site.

The Kcr-specific affinity reagent may specifically bind to a mouse H1.2histone protein (SEQ ID NO: 6) or a fragment thereof when the mouse H1.2histone protein or a fragment thereof is crotonylated at a crotonylationsite selected from the group consisting of mouse H1.2K33, H1.2K63,H1.2K84, H1.2K158 and H1.2K167, but not when the H1.2 histone protein ora fragment thereof is not crotonylated at the crotonylation site.

The Kcr-specific affinity reagent may specifically bind to a mouse H2Ahistone protein (SEQ ID NO: 7) or a fragment thereof when the mouse H2Ahistone protein or a fragment thereof is crotonylated at a crotonylationsite selected from the group consisting of mouse H2AK36 and H2AK118, butnot when the H2A histone protein or a fragment thereof is notcrotonylated at the crotonylation site.

The Kcr-specific affinity reagent may specifically bind to a mouse H2Bhistone protein (SEQ ID NO: 8) or a fragment thereof when the mouse H2Bhistone protein or a fragment thereof is crotonylated at a crotonylationsite selected from the group consisting of mouse H2BK5, H2BK11, H2BK12,H2BK15, H2BK16, H2BK20, H2BK23 and H2BK34, but not when the H2B histoneprotein or a fragment thereof is not crotonylated at the crotonylationsite.

The Kcr-specific affinity reagent may specifically bind to a mouse H3histone protein (SEQ ID NO: 9) or a fragment thereof when the mouse H3histone protein or a fragment thereof is crotonylated at a crotonylationsite selected from the group consisting of mouse H3K4, H3K9, H3K18,H3K23, H3K27 and H3K56, but not when the H3 histone protein or afragment thereof is not crotonylated at the crotonylation site.

The Kcr-specific affinity reagent may specifically bind to a mouse H4histone protein (SEQ ID NO: 10) or a fragment thereof when the mouse H4histone protein or a fragment thereof is crotonylated at a crotonylationsite selected from the group consisting of mouse H4K5, H4K8 and H4K16,but not when the H4 histone protein or a fragment thereof is notcrotonylated at the crotonylation site.

The Kcr-specific affinity reagent may be polyclonal or monoclonalantibody. The polyclone or monoclonal antibody may be prepared usingconventional techniques known in the art. An immortalized cell line maybe prepared using conventional techniques known in the art to producethe monoclonal antibody. The monoclonal antibody may be generated byscreening of a phage library, a synthetic peptide library, or a yeastlibrary. The antibody may also be obtained from an antibody productionmethod of the present invention.

The Kcr-specific affinity reagent of the present invention may be awhole antibody. The whole antibody consists of two heavy chains and twolight chains. The heavy and light chains contain variable regions, whichcontribute to the specificity of antigen binding by the antibody. Thewhole antibody comprises an antigen binding portion. A crotonylationsite specific binding domain within a whole antibody may be determinedusing conventional techniques known in the art.

The Kcr-specific affinity reagent of the present invention may be anantigen binding portion of a whole antibody. The antigen binding portionmay specifically bind to a histone protein or a fragment thereofcomprising a crotonylation site. In some embodiments, the antigenbinding portion specifically binds to the histone protein or a fragmentthereof when the histone protein or a fragment thereof is crotonylated,and does not bind to the histone protein or a fragment thereof when thehistone protein or a fragment thereof is not crotonylated. Preferably,the antigen binding portion is crotonylation site specific. In someembodiments, the crotonylation site specific antigen binding portionspecifically binds to a histone protein or a fragment thereof comprisinga crotonylation site when the histone protein or a fragment thereof iscrotonylated at the crotonylation site, and does not bind to the histoneprotein or a fragment thereof when the histone protein or a fragmentthereof is not crotonylated at the crotonylation site.

The antigen binding portion may be obtained by using conventionaltechniques known in the art. A crotonylation site specific bindingdomain within an antigen binding portion may be determined usingconventional techniques known in the art.

The Kcr-specific affinity reagent of the present invention may be asingle-chain variable fragment that specifically binds to a protein or afragment thereof comprising a crotonylation site. In some embodiments,the single-chain variable fragment specifically binds a histone proteinor a fragment thereof when the histone protein or a fragment thereof iscrotonylated, and does not bind to the histone protein or a fragmentthereof when the histone protein or a fragment thereof is notcrotonylated.

Preferably, the single-chain variable fragment is crotonylation sitespecific. In some embodiments, the crotonylation site specificsingle-chain variable fragment specifically binds to a histone proteinor a fragment thereof comprising a crotonylation site when the histoneprotein or a fragment thereof is crotonylated at the crotonylation site,and does not bind to the histone protein or a fragment thereof when thehistone protein or a fragment thereof is not crotonylated at thecrotonylation site.

The single-chain variable fragment may be obtained as a fusion proteincomprising variable regions of the heavy and light chains of a wholeantibody, connected with a short linker peptide. The linker peptide mayhave about 1-50 amino acids, preferably about 1-30 amino acids, morepreferably about 2-15 amino acids. In particular, the variable regionsof the heavy and light chains may be determined by conventionaltechniques known in the art. A crotonylation site specific bindingdomain within a single-chain variable fragment may be determined usingconventional techniques known in the art.

A method for detecting protein crotonylation in a sample is furtherprovided. The method comprises (a) contacting the sample with aKcr-specific affinity reagent of the present invention to form a bindingcomplex, and (b) detecting the binding complex. The presence of thebinding complex indicates protein crotonylation in the sample. Thebinding complex may be detected by using various conventional methods inthe art. The protein crotonylation may be histone crotonylation. TheKcr-specific affinity reagent may specifically bind to a histone proteinor a fragment thereof comprising a crotonylation site when the histoneprotein or a fragment thereof is crotonylated.

The sample may be a biological sample (e.g., bodily fluid or serum). Thebiological sample be obtained from a subject. The subject may be amouse, rat or human. Preferably, the subject is a human, more preferablya human who has suffered from or is predisposed to a disease or disorder(e.g., cancer, neurodegenerative diseases, aging, metabolic disorder, ordysgenesis).

Any of the Kcr-specific affinity reagents of the present invention maybe used for the crotonylation detection method. Preferably, theKcr-specific affinity reagent is a crotonylation site specific antibody.More preferably, the crotonylation site specific antibody specificallybinds to a histone protein or a fragment thereof when the histoneprotein or a fragment thereof is crotonylated at the crotonylation site,and does not bind to the histone protein or a fragment thereof when thehistone protein or a fragment thereof is not crotonylated at thecrotonylation site. The histone protein may be selected from the groupconsisting of human and mouse H1.2, H2A, H2B, H3 and H4 histoneproteins. The crotonylation site may be selected from the groupconsisting of human H1.2K33, H1.2K63, H1.2K84, H1.2K89, H1.2K96,H1.2K158, H1.2K167, H2AK36, H2AK118, H2AK119, H2AK125, H2BK5, H2BK11,H2BK12, H2BK15, H2BK16, H2BK20, H2BK23, H2BK34, H3K4, H3K9, H3K18,H3K23, H3K27, H3K56, H4K5, H4K8, H4K12 and H4K16, and mouse H1.2K33,H1.2K63, H1.2K84, H1.2K158, H1.2K167, H2AK36, H2AK118, H2BK5, H2BK11,H2BK12, H2BK15, H2BK16, H2BK20, H2BK23, H2BK34, H3K4, H3K9, H3K18,H3K23, H3K27, H3K56, H4K5, H4K8 and H4K16, and homologous lysine sitesin corresponding eukaryotic histone proteins.

The Kcr-specific affinity reagents of the present invention may beuseful for detecting protein crotonylation in a sample. ExemplaryKcr-specific affinity reagents include those that specifically bind to ahuman H1.2 histone protein (SEQ ID NO: 1) when crotonylated at H1.2K33,H1.2K63, H1.2K84, H1.2K89, H1.2K96, H1.2K158 or H1.2K167; a human H2Ahistone protein (SEQ ID NO: 2) when crotonylated at H2AK36, H2AK118,H2AK119 or H2AK125; a human H2B histone protein (SEQ ID NO: 3) whencrotonylated at H2BK5, H2BK11, H2BK12, H2BK15, H2BK16, H2BK20, H2BK23 orH2BK34; a human H3 histone protein (SEQ ID NO: 4) when crotonylated atH3K4, H3K9, H3K18, H3K23, H3K27 or H3K56; a human H4 histone protein(SEQ ID NO: 5) when crotonylated at H4K5, H4K8, H4K12 or H4K16; a mouseH1.2 histone protein (SEQ ID NO: 6) when crotonylated at H1.2K33,H1.2K63, H1.2K84, H1.2K158 or H1.2K167; a mouse H2A histone protein (SEQID NO: 7) when crotonylated at H2AK36 or H2AK118; a mouse H2B histoneprotein (SEQ ID NO: 8) when crotonylated at H2BK5, H2BK11, H2BK12,H2BK15, H2BK16, H2BK20, H2BK23 or H2BK34; a mouse H3 histone protein(SEQ ID NO: 9) when crotonylated at H3K4, H3K9, H3K18, H3K23, H3K27 orH3K56; or a mouse H4 histone protein (SEQ ID NO: 10) when crotonylatedat H4K5, H4K8 or H4K16. Examples of suitable Kcr-specific affinityreagents include anti-H3K23cr rabbit pAb, anti-H3K56cr rabbit pAb,anti-crotonyl-histone H2A (Lys119) rabbit pAb, anti-crotonyl-histone H2B(Lys11) rabbit pAb, anti-crotonyl-histone H2B (Lys12) rabbit pAb,anti-crotonyl-histone H2B (Lys16) rabbit pAb, anti-crotonyl-histone H2B(Lys20) rabbit pAb, anti-crotonyl-histone H2B (Lys34) rabbit pAb,anti-crotonyl-histone H3 (Lys4) rabbit pAb, anti-crotonyl-histone H3(Lys4) mouse mAb, anti-crotonyl-histone H3 (Lys18) rabbit pAb,anti-crotonyl-histone H3 (Lys23) mouse mAb, anti-crotonyl-histone H3(Lys23) rabbit pAb, and anti-crotonyl-histone H3 (Lys27) mouse mAb.

For each protein crotonylation detection method of the presentinvention, a kit is provided. The kit comprises a Kcr-specific affinityreagent that specifically binds to a protein or a fragment thereofcomprising a crotonylation site. The kit may include an instructiondirecting how to carry out the detection method. The proteincrotonylation may be histone crotonylation. Preferably, the Kcr-specificaffinity reagent specifically binds to a protein or a fragment thereofcomprising when the protein or a fragment thereof is crotonylated at thecrotonylation site. The Kcr-specific affinity reagent may be an isolatedantibody of the present invention. Examples of suitable antibodiesinclude anti-H3K23cr rabbit pAb, anti-H3K56cr rabbit pAb,anti-crotonyl-histone H2A (Lys1119) rabbit pAb, anti-crotonyl-histoneH2B (Lys11) rabbit pAb, anti-crotonyl-histone H2B (Lys12) rabbit pAb,anti-crotonyl-histone H2B (Lys16) rabbit pAb, anti-crotonyl-histone H2B(Lys20) rabbit pAb, anti-crotonyl-histone H2B (Lys34) rabbit pAb,anti-crotonyl-histone H3 (Lys4) rabbit pAb, anti-crotonyl-histone H3(Lys4) mouse mAb, anti-crotonyl-histone H3 (Lys18) rabbit pAb,anti-crotonyl-histone H3 (Lys23) mouse mAb, anti-crotonyl-histone H3(Lys23) rabbit pAb, and anti-crotonyl-histone H3 (Lys27) mouse mAb.

A fusion protein reporter is provided. The fusion protein reportercomprises a core flanked by a donor fluorescent moiety and an acceptorfluorescent moiety. The core comprises a peptide comprising acrotonylation site, and a crotonylation binding domain.

The fusion protein reporter of the present invention is useful fordetermining the protein crotonylation level in a sample or screening foran agent that regulates protein crotonylation by using the fluorescenceresonance energy transfer (FRET). The FRET involves the transfer ofphotonic energy between fluorophores when in close proximity. Donorfluorescent moieties and acceptor fluorescent moieties suitable for FRETare known in the art. In the fusion protein reporter, the donorfluorescent moiety may be selected from the group consisting of cyanfluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), andA206K mutants thereof, and the acceptor fluorescent moiety may beselected from the group consisting of yellow fluorescent protein (YFP),enhanced yellow fluorescence protein (EYFP), Citrine, Venus, and A206Kmutants thereof.

The peptide in the fusion protein reporter may be a peptide of thepresent invention. It may be a peptide derived from a histone protein ora fragment thereof comprising the crotonylation site. It may becrotonylated or not crotonylated at the crotonylation site.

The crotonylation site may be located in the N-terminus, C-terminus orthe core region of a histone protein. The N-terminus, C-terminus, andcore regions of histone proteins (e.g., human or mouse H1.2, H2A, H2B,H3 or H4) are known in the art.

The fusion protein reporter may comprise one or more crotonylationbinding domains. A crotonylation binding domain may be derived from aKcr-specific affinity reagent that specifically binds to a histoneprotein or a fragment thereof comprising a crotonylation site when thehistone protein or a fragment thereof is crotonylated or notcrotonylated at the crotonylation site. The crotonylation binding domainmay be the crotonylation site specific binding domain within aKcr-specific affinity reagent, for example, a whole antibody, an antigenbinding portion, or a single-chain variable fragment, of the presentinvention. The crotonylation binding domain may be derived from aKcr-specific affinity reagent of the present invention.

In some embodiments, the peptide is not crotonylated at thecrotonylation site, and the crotonylation binding domain specificallybinds to the peptide when the peptide is crotonylated at thecrotonylation site, and does not bind to the peptide when the peptide isnot crotonylated at the crotonylation site. Examples of thenon-crotonylated peptides include QLATKAA, CQLATKAA, YQKST, CYQKSTELL,LLPKKTESHHKAK, CLLPKKTESHHKAKG, APAPKKGS, APAPKKGSC, CAPAPKKGS, GSKKA,GSKKAVTC, TKAQKKDG, AVTKAQKKDGC, ARTKQTAR, ARTKQTARC, APRKQLA,APRKQLATC, QLATKAARK, QLATKAARKC, AARKSAP, AARKSAPATGGC, CRLLRKGNYAER,CAVTKAQKKDG, CARTKQTARKSTG, CSGRGKGG and CGLGKGGAKRHR.

In other embodiments, the peptide is crotonylated at the crotonylationsite, and the crotonylation binding domain specifically binds to thepeptide when the peptide is not crotonylated at the crotonylation site,and does not bind to the peptide when the peptide is crotonylated at thecrotonylation site. Examples of the crotonylated peptides includeQLATKcrAA, CQLATKcrAA, YQKcrST, CYQKcrSTELL, LLPKKcrTESHHKAK,CLLPKKcrTESHHKAKG, APAPKcrKGS, APAPKcrKGSC, APAPKKcrGS, CAPAPKKcrGS,GSKKcrA, GSKKcrAVTC, TKcrAQKKDG, AVTKcrAQKKDGC, ARTKcrQTAR, ARTKcrQTARC,APRKcrQLA, APRKcrQLATC, QLATKcrAARK, QLATKcrAARKC, AARKcrSAP,AARKcrSAPATGGC, CRLLRKcrGNYAER, CLLPKcrKTESHHKAKG, CLLPKKTESHHKcrAKG,CAVTKAQKcrKDG, CARTKQTARKcrSTG, CSGRGKcrGG and CGLGKGGAKcrRHR. Thepeptide may be conjugated to the crotonylation binding domain with alinker molecule. The linker molecule may be a peptide have any aminoacid sequence, and may have about 1-50 amino acids, preferably 1-30amino acids, more preferably 2-15. In some embodiments, the linkermolecule may be -Gly-Gly-. The length and contents of a linker moleculemay be adjusted to optimize potential fluorescence resonance energytransfer (FRET) between the donor fluorescent moiety and the acceptorfluorescent moiety when the peptide in the fusion protein reporter iscrotonylated, or de-crotonylated, and bound by the crotonylation bindingdomain.

The fusion protein reporter may further comprise a targetingpolypeptide. The targeting polypeptide may be selected from the groupconsisting of a receptor ligand, a nuclear localization sequence (NLS),a nuclear export signal (NES), a plasma membrane targeting signal, ahistone binding protein, and a nuclear protein.

A method for determining the level of protein crotonylation in a sampleis provided. The method comprises (a) contacting the sample with afusion protein reporter, and (b) comparing the level of fluorescenceresonance energy transfer (FRET) between the donor fluorescent moietyand the acceptor fluorescent moiety after contacting with that beforecontacting. The fusion protein reporter comprises a core flanked by adonor fluorescent moiety and an acceptor fluorescent moiety. The corecomprises a peptide comprising a crotonylation site, and a crotonylationbinding domain. The peptide is not crotonylated at the crotonylationsite. Examples of the non-crotonylated peptides include QLATKAA,CQLATKAA, YQKST, CYQKSTELL, LLPKKTESHHKAK, CLLPKKTESHHKAKG, APAPKKGS,APAPKKGSC, CAPAPKKGS, GSKKA, GSKKAVTC, TKAQKKDG, AVTKAQKKDGC, ARTKQTAR,ARTKQTARC, APRKQLA, APRKQLATC, QLATKAARK, QLATKAARKC, AARKSAP,AARKSAPATGGC, CRLLRKGNYAER, CAVTKAQKKDG, CARTKQTARKSTG, CSGRGKGG andCGLGKGGAKRHR. The crotonylation binding domain specifically binds to thepeptide when the peptide is crotonylated at the crotonylation site, anddoes not bind to the peptide when the peptide is not crotonylated at thecrotonylation site. The level of FRET indicates the level of proteincrotonylation in the sample. The level of FRET may be increased ordecreased after contacting. The method may further comprise adding anagent to the sample, and the agent regulates protein crotonylation. Theagent may promote or inhibit protein crotonylation. The proteincrotonylation may be histone crotonylation. A fusion protein reporter ofthe present invention may be used in this method.

In the method for determining the protein crotonylation level in asample, the sample may be a biological sample. The biological sample maycomprise a cell, a tissue biopsy, or a clinical fluid. The biologicalsample may be obtained from a subject (e.g., a mouse, rat, or human).The subject is healthy. The subject may have suffered from or may bepredisposed to a histone crotonylation related disorder. A histonecrotonylation related disorder refers to a disorder or disease linked toabnormal regulation of histone crotonylation. Examples of a histonecrotonylation related disorder may include cancer, neurodegenerativediseases, aging, metabolic disorder, and dysgenesis. The method mayfurther comprise comparing the FRET level in the sample with a controlFRET level. The control FRET level may be the FRET level in a controlsample obtained from a subject, and the subject has not suffered from orpredisposed to a histone crotonylation related disorder. The FRET levelin the sample may be higher or lower than the control FRET level.

A method for determining the level of protein de-crotonylation in asample is provided. The method comprises (a) contacting the sample witha fusion protein reporter, and (b) comparing the level of fluorescenceresonance energy transfer (FRET) between the donor fluorescent moietyand the acceptor fluorescent moiety after contacting with that beforecontacting. The fusion protein reporter comprises a core flanked by adonor fluorescent moiety and an acceptor fluorescent moiety. The corecomprises a peptide comprising a crotonylation site that iscrotonylated, and a crotonylation binding domain. The peptide iscrotonylated at the crotonylation site. Examples of the crotonylatedpeptides include QLATKcrAA, CQLATKcrAA, YQKcrST, CYQKcrSTELL,LLPKKcrTESHHKAK, CLLPKKcrTESHHKAKG, APAPKcrKGS, APAPKcrKGSC, APAPKKcrGS,CAPAPKKcrGS, GSKKcrA, GSKKcrAVTC, TKcrAQKKDG, AVTKcrAQKKDGC, ARTKcrQTAR,ARTKcrQTARC, APRKcrQLA, APRKcrQLATC, QLATKcrAARK, QLATKcrAARKC,AARKcrSAP, AARKcrSAPATGGC, CRLLRKcrGNYAER, CLLPKcrKTESHHKAKG,CLLPKKTESHHKcrAKG, CAVTKAQKcrKDG, CARTKQTARKcrSTG, CSGRGKcrGG andCGLGKGGAKcrRHR. The crotonylation binding domain specifically binds tothe peptide when the peptide is not crotonylated at the crotonylationsite, and does not bind to the peptide when the peptide is crotonylatedat the crotonylation site. The level of FRET indicates the level ofprotein de-crotonylation in the sample. The level of FRET may beincreased or decreased after contacting. The method may further compriseadding an agent to the sample, and the agent regulates proteinde-crotonylation. The agent may promote or inhibit proteincrotonylation. Protein de-crotonylation may be histone de-crotonylation.A fusion protein reporter of the present invention may be used in thismethod.

In the method for determining the protein de-crotonylation level in asample, the sample may be a biological sample. The biological sample maycomprise a cell, a tissue biopsy, or a clinical fluid. The biologicalsample may be obtained from a subject (e.g., a mouse, rat or human). Thesubject is healthy. The subject may have suffered from or may bepredisposed to a histone de-crotonylation related disorder. A histonede-crotonylation related disorder refers to a disorder or disease linkedto abnormal regulation of histone crotonylation. Examples of a histonede-crotonylation related disorder may include cancer, neurodegenerativediseases, aging, metabolic disorder, and dysgenesis. The method mayfurther comprise comparing the FRET level in the sample with a controlFRET level. The control FRET level may be the FRET level in a controlsample obtained from a subject, and the subject has not suffered from orpredisposed to a histone de-crotonylation related disorder. The FRETlevel in the sample may be higher or lower than the control FRET level.

A method for screening an agent that regulates protein crotonylation isprovided. The method comprises (a) contacting a candidate agent with afusion protein reporter, and (b) comparing the level of fluorescenceresonance energy transfer (FRET) between the donor fluorescent moietyand the acceptor fluorescent moiety after contacting with that beforecontacting. The fusion protein reporter comprises a core flanked by adonor fluorescent moiety and an acceptor fluorescent moiety. The corecomprises a peptide comprising a crotonylation site, and a crotonylationbinding domain. The peptide is not crotonylated at the crotonylationsite. Examples of the non-crotonylated peptides include QLATKAA,CQLATKAA, YQKST, CYQKSTELL, LLPKKTESHHKAK, CLLPKKTESHHKAKG, APAPKKGS,APAPKKGSC, CAPAPKKGS, GSKKA, GSKKAVTC, TKAQKKDG, AVTKAQKKDGC, ARTKQTAR,ARTKQTARC, APRKQLA, APRKQLATC, QLATKAARK, QLATKAARKC, AARKSAP,AARKSAPATGGC, CRLLRKGNYAER, CAVTKAQKKDG, CARTKQTARKSTG, CSGRGKGG andCGLGKGGAKRHR. The crotonylation binding domain specifically binds to thepeptide when the peptide is crotonylated at the crotonylation site, anddoes not bind to the peptide when the peptide is not crotonylated at thecrotonylation site. An increase in the level of FRET after contactingindicates that the candidate agent promotes protein crotonylation whilea decrease in the level of FRET after contacting indicates that thecandidate agent inhibits protein crotonylation. The candidate agent maybe a compound or a biological molecule. The protein crotonylation may behistone crotonylation.

A method for screening an agent that regulates protein de-crotonylationis provided. The method comprises (a) contacting a candidate agent witha fusion protein reporter, and (b) comparing the level of fluorescenceresonance energy transfer (FRET) between the donor fluorescent moietyand the acceptor fluorescent moiety after contacting with that beforecontacting. The fusion protein reporter comprises a core flanked by adonor fluorescent moiety and an acceptor fluorescent moiety. The corecomprises a peptide comprising a crotonylation site that iscrotonylated, and a crotonylation binding domain. The peptide iscrotonylated at the crotonylation site. Examples of the crotonylatedpeptides include QLATKcrAA, CQLATKcrAA, YQKcrST, CYQKcrSTELL,LLPKKcrTESHHKAK, CLLPKKcrTESHHKAKG, APAPKcrKGS, APAPKcrKGSC, APAPKKcrGS,CAPAPKKcrGS, GSKKcrA, GSKKcrAVTC, TKcrAQKKDG, AVTKcrAQKKDGC, ARTKcrQTAR,ARTKcrQTARC, APRKcrQLA, APRKcrQLATC, QLATKcrAARK, QLATKcrAARKC,AARKcrSAP, AARKcrSAPATGGC, CRLLRKcrGNYAER, CLLPKcrKTESHHKAKG,CLLPKKTESHHKcrAKG, CAVTKAQKcrKDG, CARTKQTARKcrSTG, CSGRGKcrGG andCGLGKGGAKcrRHR. The crotonylation binding domain specifically binds tothe peptide when the peptide is not crotonylated at the crotonylationsite, and does not bind to the peptide when the peptide is crotonylatedat the crotonylation site. An increase in the level of FRET aftercontacting indicates that the candidate agent promotes proteinde-crotonylation while a decrease in the level of FRET after contactingindicates that the candidate agent inhibits protein de-crotonylation.The candidate agent may be a compound or a biological molecule. Proteinde-crotonylation may be histone de-crotonylation.

A method for treating or preventing a protein crotonylation relateddisease in a subject in need thereof is provided. The method comprisesadministering to the subject an effective amount of a compositioncomprising an agent that regulates protein crotonylation. The agent maybe identified by the screening method of the present invention. Theprotein crotonylation may be histone crotonylation.

A method for treating or preventing a protein de-crotonylation relateddisease in a subject in need thereof is provided. The method comprisesadministering to the subject an effective amount of a compositioncomprising an agent that regulates protein de-crotonylation. The agentmay be identified by the screening method of the present invention.Protein de-crotonylation may be histone de-crotonylation.

Example 1 Materials

All peptides used in this study were synthesized through customersynthesis using Fmoc-Lysine (crotonyl)-OH. All chemicals of the highestpurity available or analytical grade and Flag M2 antibody were purchasedfrom Sigma-Aldrich, Inc. (St. Louis, Mo.). HA antibody was purchasedfrom Roche Diagnostics (Indianapolis, Ind.). The histones were extractedfrom S. cerevisiae cells, S2 cells, mouse embolic fibroblast (MEF)cells, human Caucasian fetal lung fibroblast (IMR90) cells, and HeLacells using previously known procedures (Shechter et al., 2007; Tateishiet al., 2009). 4,4,4,3-D4-crotonic acid was prepared usingD4-acetaldehyde (Cambridge Isotope Laboratories, Andover, Mass.) andmalonic acid. Polyclonal pan anti-Kcr and anti-Kac antibodies weregenerated in house using a procedure described below.

Preparation of Histones from HeLa Cells

The process of preparing HeLa cell histones were known (Zhang et al.,2010). HeLa cells were grown in DMEM culture medium supplemented with10% fetal bovine serum. The cells were then harvested and washed twicewith ice-cold PBS containing 5 mM sodium butyrate. The cells were lysedin Triton extraction buffer (TEB; PBS containing 0.5% (v/v) TritonX-100, 2 mM PMSF, and 0.02% (w/v) NaN3). After centrifugation, thesupernatant was removed. The pellet was washed, centrifuged, andresuspended in 0.4 N H2SO4 overnight at 4° C. After centrifugation, thesupernatant was removed; histones in the supernatant were precipitatedby the addition of 20% (v/v, final concentration) TCA to the proteinsolution. The suspension was incubated at −20° C. for 4 hrs. The proteinprecipitate was spun down, collected, and washed with acidified acetone(0.1% (v/v) HCl), followed by two washes with ice-cold acetone. Afterbeing dried at room temperature, the pellets were dissolved in water.

In-Solution Proteolytic Digestion and Chemical Derivatization of HistoneProteins

In-solution tryptic digestion of histone samples was carried out using aknown protocol (Kim et al., 2006; Luo et al., 2008). In vitro lysinepropionylation of histone extract and tryptic histone peptides wasperformed as known in the art (Garcia et al., 2007a). Three differentprocesses of proteolytic digestion were performed: histone extracts were(i) in-solution digested without chemical propionylation, (ii)chemically propionylated after in-solution digestion, or (iii)chemically propionylated before in-solution digestion.

Isoelectric focusing (IEF) fractionation

The histone proteolytic peptides were separated using an Agilent 3100OFFGEL Fractionator (Agilent, Santa Clara, Calif.) according to themanufacturer's instructions and generally known in the art. Twelvefractions were obtained from each IEF fractionation experiment.

Synthesis of Bovine Serum Albumin (BSA) Derivatives

Five mg of but-3-enonylic acid, crotonic acid, or metharylic acid wasmixed with 5 mg of BSA in 4 ml of PBS buffer, followed by the additionof 25 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Themixture was stirred at room temperature for 4 hrs to generatevinylacetyl-BSA, crotonyl-BSA, and methacryl-BSA, respectively. Theunreacted EDC and other small molecules were removed from BSAderivatives by gel filtration. The modified BSAs were confirmed bySDS-PAGE.

Conjugation of Crotonyllysine-Immobilized Agarose Beads

The crotonyllysine residue was conjugated to AminoLink Plus CouplingResin (Pierce Biotechnology, Rockford, Ill.) following themanufacturer's protocol. Two mL of resin were washed with PBS and thensuspended in 6 mL PBS. The beads were then mixed with 2 mg of thecrotonylysine (pre-solubilized in 2 ml PBS) and then NaCNBH3 (to a finalconcentration of 50 mM) was added. After incubation for 6 hrs at roomtemperature with agitation, the beads were washed by 4 ml of PBS andthen blocked by 2 ml of 1.0 M Tris.HCl, pH 7.4 for 30 min at roomtemperature. The beads were sequentially washed with 10 mL of 1.0 M NaCland 4 ml of PBS.

Generation of Pan Anti-Crotonyllysine Antibodies

The anti-crotonyllysine IgG was developed by immunizing 10 rabbits withlysine-crotonylated BSA. The rabbits were immunized with fourinjections. Five batches of serums were collected from each rabbit. Theserum with the highest ELSA titer was used for enrichinganti-crotonyllysine antibody.

The pan anti-crotonyllysine antibody was enriched using thecrotonyllysine-conjugate agarose beads. About 10 mL of serum wereincubated overnight with 2 mL of the crotonyllysine-conjugated agarosebeads in a column. The beads were then sequentially washed with 20 mL ofPBSN buffer (PBS containing 0.5% NP40), 20 mL of PBSS buffer (PBScontaining 0.1% SDS), 6 mL of PBSS (PBS containing 0.8 M NaCl), and 6 mLof PBS. The bound antibodies were eluted from the beads with 0.1 Mglycine (pH 3.0) and immediately neutralized with 1.0 M Tris-HCl (pH8.5). The antibodies were dialyzed against in cold PBS overnight. Bothdot-spot assay and Western blotting were performed to check quality ofthe antibody.

The pan anti-Kac antibodies were developed likewise usinglysine-acetylated BSA as an antigen. The antibody was purified usingacetyllysine-conjugated agarose by the above procedure.

FIG. 7 is the Western blot result showing the existence of lysinecrotonylation in HeLa cell lysates with pan anti-Kcr antibody. HeLacells were cultured in DMEM medium with/without crotonate (50 mM) for 12hrs. Cells were lysed with sampling buffer. The protein from whole celllysates were resolved on SDS-PAGE and Western blotting by anti-Kcrantibody.

Generation of Sequence-Specific Anti-Crotonyllysine Antibodies

As a particular embodiment, a sequence-specific anti-H3K23crotonyllysine antibody (anti-H3K23cr rabbit pAb) was developed byimmunizing rabbits with an antigen peptide bearing a crotonyllysineresidue, CQLATKAA, where C is a cystein residue, and the underlined Kindicates crotonyllysine residue. The rabbits were immunized with fourinjections. Five batches of serums were collected from each rabbit. Theserum with the highest ELSA titer was used for enrichingsequence-specific anti-crotonyllysine antibodies.

The sequence-specific anti-crotonyllysine antibodies was enriched usingthe antigen-conjugated agarose beads. The serums were centrifuged at20,000 g to remove possible protein particles. About 10 mL of serum wereincubated overnight with 2 mL of the crotonyllysine-containing peptideantigen conjugated agarose beads in a column. The beads were thensequentially washed with 20 mL of PBSN buffer (PBS containing 0.5%NP40), and 6 mL of PBS. The bound antibodies were eluted from the beadswith 0.1 M glycine (pH 3.0) and immediately neutralized with 1.0 MTris-HCl (pH 8.5). The antibodies were dialyzed against in cold PBSovernight. The obtained antibodies were depleted by incubating with theagarose conjugated with a peptide, CQLATKAA, which has the same peptidesequence as the antigen peptide, but the lysine residue is notcrotonylated. As it would be understood by a person with ordinary skillin the art, other antigen peptides other than the one used in thisparticular embodiment, i.e., CQLATKAA, may also be used to obtainsatisfactory results. The design of the antigen peptide is based on thesequence around a lysine in the protein whose crotonylation is intendedas the target of detection and requires only ordinary kill in the art.

With the same method, another antigen peptide CYQKSTELL (the underlinedK is a lysine crotonylated), was used to generate sequence specificantibodies for H3K56 lysine crotonylation (anti-H3K56cr rabbit pAb). Theprocesses are the same as described in the above except that andifferent antigen peptide (i.e., CYQKSTELL) was used for targetingH3K56, that is, lysine crotonylation at position 56 of the H3 protein.

FIG. 8 shows the detection of H3K23 and H3K56 crotonylation by Westernblotting using sequence-specific antibodies. Human HeLa histones wereseparated by SDS-PAGE and Western blotted by H3K23 crotonyllysine sitespecific antibody (anti-H3K23cr rabbit pAb) or H3K56 crotonyllysine sitespecific antibody (anti-H3K56cr rabbit pAb) competed by nonmodified (K)or crotonyllysine (Kcr) sequence specific peptide.

While in the above embodiments anti-crotonyllysine IgG was used, peoplewith ordinary skill of the art may practice the present invention withcorresponding monoclonal antibodies or single-chain variable fragments(scFvs) to obtain a satisfactory result.

Affinity Enrichment of Crotonyllysine Peptides Using Anti-Kcr Antibody

The affinity-purified anti-crotonyllysine antibody was immobilized toprotein A agarose beads (GE Healthcare Biosciences, Pittsburgh, Pa.) byincubation at 4° C. for 4 hrs. The supernatant was removed and the beadswere washed three times with NETN buffer (50 mM Tris.HCl [pH 8.0], 100mM NaCl, 1 mM EDTA, 0.5% NP40).

The histone tryptic peptides were resolubilized in NETN buffer. Affinitypurification was carried out by incubating the peptides with 20 μl ofanti-crotonyllysine antibody-immobilized protein A beads at 4° C.overnight with gentle shaking. The beads were washed three times withNETN buffer and twice with ETN buffer (50 mM Tris-HCl pH 8.0, 100 mMNaCl, 1 mM EDTA). The bound peptides were eluted from the beads bywashing three times with 50 μl of 0.1% TFA. The elutes were combined anddried in a SpeedVac.

Western Blotting with Competition with a Peptide Library

One μg of histone protein extracts were resolved in SDS-PAGE.Crotonylation signal was detected by pan anti-crotonyllysine antibodywith competition by a peptide library bearing a non-modified, acetyl,propionyl, butyryl, methacrylyl, or crotonyl lysine.

In-Solution Proteolytic Digestion and Chemical Derivatization of HistoneProteins

Histone tryptic peptides were generated by three methods: (i) Generationof histone peptides without in-vitro lysine propionylation. The histonepellet obtained above was suspended in 50 mM ammonium bicarbonatesolution (pH 8.4) and was digested using a protocol previously described(Kim et al., 2006; Luo et al., 2008); (ii) In vitro lysinepropionylation after histone tryptic digestion. The in vitro chemicalreaction was performed as previously described (Garcia et al., 2007b).To generate derivatized histone peptides, 3 mg of histone trypticdigests obtained above were dissolved in 25 μl of 100 mM ammoniumbicarbonate buffer (pH 8.0), and 600 μl of 50% propionic anhydride inmethanol (v/v) was added into the solution. The pH of the solution wasquickly adjusted to pH 8.0 with ammonium hydroxide. The mixture was thenincubated at 51° C. for 20 min and dried in a SpeedVac. The procedurewas repeated once to ensure completion of the chemical reaction. (iii)In vitro lysine propionylation of core histones prior to trypticdigestion. Histones were derivatized by propionylation reaction asdescribed above, and the derivatized histones were subjected toin-solution tryptic digestion overnight.

HPLC/MS/MS Analysis and Protein Sequence Database Searching

The dried peptide extracts were dissolved in 3 μl HPLC solvent A (0.1%formic acid in water, v/v). 1 μl sample was injected into a NanoLC-1Dplus HPLC system (Eksigent Technologies, Dublin, Calif.), which wasconnected to a home-made capillary Jupiter C12 column (10 cm length×75μm ID, 4 μm particle size, 90 Å pore size; Phenomenex, St. Torrance,Calif.). Peptides were eluted with a 2-hour gradient of 2% to 80% HPLCsolvent B (0.1% formic acid in acetonitrile, v/v) in solvent A at a flowrate of 200 nl/min. Peptides were then ionized and analyzed by LTQOrbitrap Velos mass spectrometer (ThermoFisher Scientific, San Jose,Calif.) using a nano-spray source. High-resolution full scan MS spectra(from m/z 350-1400) were acquired in the Orbitrap with resolutionR=60,000 at m/z 400 and lockmass enabled (m/z at 445.120025), followedby MS/MS fragmentation of the twenty most intense ions in the linear iontrap with collisionally activated dissociation (CAD) energy of 35%. Theexclusion duration for the data-dependent scan was 36 seconds, and theexclusion window was set at ±0.01% m/z.

The MS/MS data were analyzed by both non-restrictive sequence alignmentby PTMap algorithm (Chen et al., 2009) and sequence alignment usinglimited, pre-specified PTMs by Mascot algorithm. The specific parametersfor protein sequence database searching included lysine mono-, di- andtri-methylation, formylation and acetylation, arginine mono-methylationand di-methylation, tyrosine hydroxylation, methionine oxidation, andlysine crotonylation (K+68.02621 Da) as variable modifications fornon-propionylated histones. For histone samples generated by trypticdigestion of propionylated histones, the specific parameters includedlysine propionylmethylation (+70.04187 Da) and lysine propionylation asvariable modifications. For histone samples propionylated after trypsindigestion, N-terminal propionylation was included as a fixedmodification. Other parameters used in data analysis were: six allowedmissing cleavages; mass error of 10 ppm for precursor ions, and 0.6 Dafor fragment ions. Charge states of +1, +2, and +3 were considered forparent ions. If more than one spectrum was assigned to a peptide, onlythe spectrum with the highest Mascot or PTMap score was selected formanual analysis. All peptides identified with peptide scores ofPTMap >0.8 and Mascot >20 were manually examined using rules known inthe art (Chen et al., 2005).

Verification of Lysine Crotonylated Peptides by HPLC/MS/MS Analysis

The lysine crotonylated peptide in tryptic digest of histones, itssynthetic counterpart, and their mixture were injected into nano-HPLCsystem and analyzed by high-resolution MS and MS/MS in the Orbitrap massspectrometer, respectively. Full MS scans were acquired with resolutionR=30,000 at m/z 400 with lockmass enabled (m/z at 445.120025), andtargeted MS/MS spectra were acquired at a resolution of 7,500 at m/z400.

Identification of Kcr Peptides

Histone proteins have a high ratio of both lysine and arginine residues.Thus, many histone tryptic peptides are relatively small andhydrophilic, some of which cannot be retained in a C18 RP-HPLC columnfor subsequent detection by MS. This problem can be addressed bychemical derivatization (e.g., lysine propionylation) of amine groups inthe protein (N-terminal amines, and free and monomethylated lysineε-amino groups) before or after tryptic digestion. Similarly, lysinepropionylation of core histones, before or after tryptic digestion, willgenerate complementary peptide sequences that boost the sequencecoverage of peptide mapping by MS. Additionally, IEF separation oftryptic digest into 12 fractions will further reduce peptide complexityand improve dynamic range.

In the present invention, as a particular embodiment, the integratedapproach was designed for systematic analysis of histone PTMs (i.e.,post-translational modifications). The strategy and results foridentifying histone PTM sites are shown in FIG. 1, which represented aneffort to maximize both the sequence coverage and sensitivity, and toidentify novel PTM sites. In this invention, MS analysis was carried outin histone proteolytic peptides that were generated by four parallelmethods (see FIG. 1A): Histone extracts were in-solution trypticdigestion without chemical propionylation (Method I), chemicallypropionylated after in-solution tryptic digestion (Method II),chemically propionylated before in-solution tryptic digestion (MethodIII), and in-gel digested after SDS-PAGE gel separation. Samples fromMethods I and II were further subjected to IEF fractionation to generate12 fractions. FIG. 1B shows peptide sequence coverage of linker and corehistones detected by the four methods. The PTM sites identified in thework as a particular embodiment of the invention are summarized in FIG.1C, where abbreviations are Kme for lysine monomethylation; Kme2 forlysine dimethylation; Kfo for lysine formylation; Kac for lysineacetylation; Rme for arginine monomethylation; Yoh for tyrosinehydroxylation; and Kcr for lysine crotonylation

PTMap, an algorithm capable of identifying all possible PTMs of aprotein (Chen et al., 2009), was used to analyze all the acquired MS/MSdata to identify histone peptides with or without a PTM. As anticipated,sequence coverage by MS mapping was significantly improved after invitro propionylation, either before or after tryptic digestion (see FIG.1B). Among the four methods, Method III (in vitro propionylation beforetryptic digestion of histones) achieved the highest sequence coverage ofhistones H1.2 (100%), H2A (90.7%) and H2B (94.4%). Method IV gave thebest coverage for histones H3 (87.3%) and H4 (82.3%). In aggregate, weachieved sequence coverage of 100% of H1.2, 90.7% of H2A, 100% of H2B,91% of H3, and 87.3% of H4. To our knowledge, this represents thehighest reported sequence coverage for peptide mapping in histones.

Using this approach, 130 unique PTM sites, including 28 Kcr sites(crotonylated lysine residue) were identified in the present invention.The remaining 102 non-Kcr modifications consist of 39 novel PTM sites,including 18 lysine monomethylation (Kme) sites, 1 lysine dimethylation(Kme2) site, 4 lysine formylation (Kfo) sites, 2 lysine acetylation(Kac) sites, 8 arginine monomethylation (Rme) sites and 6 tyrosinehydroxylation (Yoh) sites (see FIG. 1C).

A summary of the non-Kcr modification sites and Kcr sites identified inthis study are shown in FIGS. 1D and 1E (where Kcr sites areunderlined), respectively. All the MS/MS spectra for the identifiedhistone PTM peptides were carefully verified as previously reported(Chen et al., 2005). It is confirmed the identification of Kcr peptidesand 10 novel non-Kcr PTM sites by MS/MS of their corresponding syntheticpeptides or by high-resolution MS/MS.

Identification of Kcr Residues in Histones

A PTM will induce structural and compositional changes in the substrateresidue and therefore a change of its molecular weight. In the presentinvention, the analysis identified, on 28 lysine residues of the corehistone peptides, a mass shift of +68 Da, which does not match the shiftassociated with any known PTM (see FIG. 1E), indicating a possiblehistone mark unknown previously.

To reveal the structure of this modification, one of these peptides,PEPAK+68SAPAPK (modified at H2BK5), was selected for further analysis.After manual inspection of the high-resolution MS data (precursor ionmass at m/z 580.8181) of this peptide, we determined the accurate massshift of this modification as +68.0230 Da. By setting the mass toleranceto ±0.01 Da (˜9 ppm, which is within the mass accuracy of the massspectrometer used), and specifying a maximum of 2 nitrogen atoms, it wasdeduced, based on the mass shift, that the possible element compositionsof the modification group as either C4H4O or H6NO3. The former, C4H5O(mass shift plus one proton), is the only reasonable molecular formulaof this modification. There were 4 possible structures consistent withthe element composition: Kcr (FIGS. 2A and 2B), vinylacetyllysine(3-butenoyllysine), methacryllysine, and cyclopropanecarboxyllysine(FIG. 2C). As crotonyl-CoA is an important and abundant intermediate(FIG. 2D), in metabolic pathways of butyryl-CoA and acetyl-CoA, Kcr wasconsidered as a putative PTM candidate. FIG. 2A shows the chemicalstructures and an illustration of the enzymatic reactions for lysineacetylation by lysine acetyltransferases (KATs) using acetyl-CoA as acofactor and the hypothesized mechanism for Kcr using crotonyl-CoA as acofactor. In FIG. 2B, the ball-and-stick models of a crotonyl group andan acetyl group are shown. The three-dimensional arrangement of fourcarbons and one oxygen of the crotonyl group that are rigid and islocated in the same plane (left). The two olefinic carbons of thecrotonyl group are shown in the middle. In contrast, the tetrahedral CH3in the acetyl group (right) can be rotated that is structurally verydifferent from the crotonyl group.

MS/MS of Synthetic Peptides and HPLC Coelution

To test if the identified mass shift of +68.0230 Da was caused by Kcr,we synthesized the Kcr peptide, PEPAKcrSAPAPK, and compared its MS/MSspectrum with that of the in vivo-derived peptide. The in vivo modifiedpeptide bearing a lysine residue with a mass shift of +68.0230 Da, thesynthetic Kcr peptide with the same peptide sequence (PEPAKcrSAPAPK),and the mixture of the two peptides exhibited almost identical parentmasses and high-resolution MS/MS spectra (FIGS. 3A to 3C). In addition,the mixture of the in vivo and synthetic peptides coeluted in HPLC/MSanalysis (FIG. 3D). These results indicated that the identified massshift of +68.0230 Da was very likely caused by Kcr.

Confirmation of Kcr Proteins by Western Blotting and Immunostaining

To further confirm Kcr in histones, a pan antibody was generated againstKcr. This pan anti-Kcr antibody specifically recognized a peptidelibrary bearing Kcr, but not four other peptide libraries in which thefixed lysine residue was unmodified (K), acetylated (Kac), propionylated(Kpr), or butyrylated (Kbu) (FIG. 4A). The specificity of the pananti-Kcr antibody was also shown by Western blotting with three bovineserum albumin (BSA) derivatives, whose lysines were chemically modifiedby a crotonyl, vinylacetyl or methacryl group, respectively. The resultshowed that pan anti-Kcr antibody only recognized the lysinecrotonylated BSA, but not the unmodified, lysine vinylacetylated orlysine methacrylated BSA (FIG. 4B). This pan anti-Kcr antibody was usedfor Western blotting and immunostaining of Kcr signal.

The antibody could detect a Kcr signal among all core histone proteins,H2A, H2B, H3, H4, and linker histone H1. In each protein, the signalcould be efficiently competed away by a peptide library bearing a Kcr,but not the peptide library bearing an unmodified lysine (FIG. 4C),metharcryllysine (FIG. 4C), acetyllysine, propionyllysine, orbutyryllysine (FIG. 4D).

By independent confirmation based on five different methods, MS/MS andHPLC coelution of synthetic peptides, D4-crotonate labeling, Westernblotting, and immunostaining, the present invention conclusivelyverified the existence of histone Kcr.

Confirmation of Kr Proteins by In Vivo D4-Crotonate Isotopic Labeling

FIG. 5A shows the dynamics of histone Kcr in response to crotonate. Thehistone proteins extracted from human prostate cancer cell line Du145incubated with 0, 50 or 100 mM crotonate for 24 hrs, were Westernblotted with anti-Kcr pan antibody. FIG. 5B shows MS/MS spectrum of PEPAKD4-crSAPAPK identified from D4-crotonate-labeled sample. The mixture ofD4-, D3- and D2-crotonyl groups was used for the identification ofD4-crotonyl peptide.

Detection of Histone Kcr as in Different Cell Types

Using the method of the present invention, it is further verified thatlysine crotonylation is present in histones from other eukaryotic cells.For example, Kcr signals were detected among core histones in samplesfrom yeast S. cerevisiae, Drosophila S2 cells, mouse embryonicfibroblast (MEF) cells, as well as human HeLa cells (FIG. 6). Takingadvantage of affinity enrichment using the pan anti-Kcr antibody andHPLC/MS/MS, 24 Kcr sites were identified on mouse MEF cells. Theresults, therefore, revealed that Kcr is an evolutionarily conservedhistone mark in eukaryotic cells.

The present invention provides an integrated approach for the systematicanalysis of histone PTMs. With this unique approach, 130 PTM sites onhuman histones, including 63 known and 67 novel histone marks wereidentified as a particular embodiment of the invention, in which Yoh andKcr were identified as two novel types of histone PTM. Therefore, thepresent invention has extended the catalogue of histone PTM sites inmammalian cells and provides a platform for the discovery of novelmechanisms of histone regulation and new ways of treating diseasesrelated to histone regulation.

Example 2

Synthetic crotonylated peptides have been designed as peptide antigensto produce histone crotonylation site specific antibodies thatspecifically recognize histone proteins when crotonylated at specificcrotonylation sites. The synthetic crotonylated peptide may have asequence selected from SEQ ID NOs: 11-43, and may have modifications onadjacent residues as shown in Table 2. Where an adjacent residue ismodified, such modification is protected. Analysis of some exemplaryhistone crotonylation site specific antibodies is described below.

Anti-crotonyl-histone H2A (Lys 119) rabbit pAb was produced byimmunizing rabbits with a synthetic crotonyl peptide, CLLPKKcrTESHHKAKG,corresponding to residues surrounding Lys 19 of human histone H2A. Theantibody was purified by protein A-conjugated agarose followed bycrotonylated histone H2A (Lys119) peptide affinity chromatography. Thisantibody detects histone H2A only when it is crotonylated at Lys119, andselectively recognizes crotonylated H2A peptide at Lys119, but not thecrotonylated H2A peptide at Lys1118 or the unmodified peptide (FIG.11A).

Anti-crotonyl-histone H2B (Lys 1) rabbit pAb was produced by immunizingrabbits with a synthetic crotonyl peptide, APAPKcrKGSC, corresponding toresidues surrounding Lys 1 of human histone H2B. The antibody waspurified by protein A-conjugated agarose followed by crotonylatedhistone H2B (Lys11) peptide affinity chromatography. This antibodydetects histone H2B only when it is crotonylated at Lys11, andselectively recognizes crotonylated H2B peptide at Lys11, but not thecrotonylated H2B peptide at Lys12 or the unmodified peptide (FIG. 12A).

Anti-crotonyl-histone H2B (Lys12) rabbit pAb was produced by immunizingrabbits with a synthetic crotonyl peptide, CAPAPKKcrGS, corresponding toresidues surrounding Lys12 of human histone H2B. The antibody waspurified by protein A-conjugated agarose followed by crotonylatedhistone H2B (Lys12) peptide affinity chromatography. This antibodydetects histone H2B only when it is crotonylated at Lys12, andselectively recognizes crotonylated H2B peptide at Lys12, but not thecrotonylated H2B peptide at Lys11 or the unmodified peptide (FIG. 13A).

Anti-crotonyl-histone H2B (Lys16) rabbit pAb was produced by immunizingrabbits with a synthetic crotonyl peptide, GSKKcrAVTC, corresponding toresidues surrounding Lys 16 of human histone H2B. The antibody waspurified by protein A-conjugated agarose followed by crotonylatedhistone H2B (Lys 16) peptide affinity chromatography. This antibodydetects histone H2B only when it is crotonylated at Lys16, andselectively recognizes crotonylated H2B peptide at Lys16, but not theunmodified peptide (FIG. 14A).

Anti-crotonyl-histone H2B (Lys20) rabbit pAb was produced by immunizingrabbits with a synthetic crotonyl peptide, AVTKcrAQKKDGC, correspondingto residues surrounding Lys20 of human histone H2B. The antibody waspurified by protein A-conjugated agarose followed by crotonylatedhistone H2B (Lys20) peptide affinity chromatography. This antibodydetects histone H2B only when it is crotonylated at Lys20, andselectively recognize crotonylated H2B peptide at Lys20, but with littlecross-reaction to the butyrylated H2B peptide at Lys20, crotonylated H2Bpeptide at Lys 23 and the unmodified peptide (FIG. 15A).

Anti-crotonyl-histone H2B (Lys34) rabbit pAb was produced by immunizingrabbits with a synthetic crotonyl peptide, CRSRKcrESY, corresponding toresidues surrounding Lys34 of human histone H2B. The antibody waspurified by protein A-conjugated agarose followed by crotonylatedhistone H2B (Lys34) peptide affinity chromatography. This antibodydetects histone H2B only when it is crotonylated at Lys34, andselectively recognize crotonylated H2B peptide at Lys34, but not thesuccinylated H2B peptide at Lys34 and the unmodified peptide (FIG. 16A).

Anti-crotonyl-histone H3 (Lys4) rabbit pAb was produced by immunizingrabbits with a synthetic crotonyl peptide, ARTKcrQTARC, corresponding toresidues surrounding Lys4 of human histone H3. The antibody was purifiedby protein A-conjugated agarose followed by crotonylated histone H3(Lys4) peptide affinity chromatography. This antibody detects histone H3only when it is crotonylated at Lys4, and selectively recognizescrotonylated H3 peptide at Lys4, but not the crotonylated or butyrylatedH3 peptide at Lys9 or the unmodified peptide (FIG. 17A).

Anti-crotonyl-histone H3 (Lys4) mouse mAb was produced by immunizingmice with a synthetic crotonyl peptide, ARTKcrQTARC, corresponding toresidues surrounding Lys4 of human histone H3. The antibody was purifiedby protein G-conjugated agarose followed by crotonylated histone H3(Lys4) peptide affinity chromatography. This antibody detects histone H3only when it is crotonylated at Lys4, and selectively recognizescrotonylated H3 peptide at Lys4, but not the crotonylated or butyrylatedH3 peptide at Lys9 or the unmodified peptide (FIG. 18A).

Anti-crotonyl-histone H3 (Lys18) rabbit pAb was produced by immunizingrabbit with a synthetic crotonyl peptide, APRKcrQLATC, corresponding toresidues surrounding Lys 18 of human histone H3. The antibody waspurified by protein A-conjugated agarose followed by crotonylatedhistone H3 (Lys 18) peptide affinity chromatography. This antibodydetects histone H3 only when it is crotonylated at Lys18, andselectively recognizes crotonylated H3 peptide at Lys18, but not theunmodified peptide or the structurally similar butyrylated orpropionylated H3 peptide at Lys 18 (FIG. 19A).

Anti-crotonyl-histone H3 (Lys23) mouse mAb was produced by immunizingmice with a synthetic crotonyl peptide, QLATKcrAARKC, corresponding toresidues surrounding Lys23 of human histone H3. The antibody waspurified by protein G-conjugated agarose followed by crotonylatedhistone H3 (Lys23) peptide affinity chromatography. This antibodydetects histone H3 only when it is crotonylated at Lys23, andselectively recognizes crotonylated H3 peptide at Lys23, but not thestructurally similar acetylated, propionylated or butyrylated H3 peptideat Lys23 (FIG. 20A).

Anti-crotonyl-histone H3 (Lys23) rabbit pAb was produced by immunizingrabbits with a synthetic crotonyl peptide, QLATKcrAARKC, correspondingto residues surrounding Lys23 of human histone H3. Antibodies arepurified by protein A-conjugated agarose followed by crotonylatedhistone H3 (Lys23) peptide affinity chromatography. The antibody waspurified with protein A-conjugated agarose followed by crotonylatedhistone H3 (Lys23) peptide affinity chromatography. This antibodydetects histone H3 only when it is crotonylated at Lys23, andselectively recognizes crotonylated H3 peptide at Lys23, but not theunmodified peptide and the structurally similar butyrylated orpropionylated H3 peptide at Lys23 (FIG. 21A).

Anti-crotonyl-histone H3 (Lys27) mouse mAb was produced by immunizingmice with a synthetic crotonyl peptide, AARKcrSAPATGGC, corresponding toresidues surrounding Lys27 of human histone H3. The antibody waspurified with protein G-conjugated agarose followed by crotonylatedhistone H3 (Lys27) peptide affinity chromatography. This antibodydetects histone H3 only when it is crotonylated at Lys27, andselectively recognizes crotonylated H3 peptide at Lys27, but not thestructurally similar acetylated peptide at Lys27 or the unmodifiedpeptide (FIG. 22A).

The term “about” as used herein when referring to a measurable valuesuch as an amount, a percentage, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate.

All documents, books, manuals, papers, patents, published patentapplications, guides, abstracts, and other references cited herein areincorporated by reference in their entirety. Other embodiments of theinvention will be apparent to those skilled in the art fromconsideration of the specification and practice of the inventiondisclosed herein. It is intended that the specification and examples beconsidered as exemplary only, with the true scope and spirit of theinvention being indicated by the following claims.

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TABLE 1  List of Histone Peptides SEQ Crotony- Peptide ID Crotonylatedlation Sequence NO Peptide Site QLATKAA 11 QLATKcrAA H3K23 CQLATKAA 12CQLATKcrAA H3K23 YQKST 13 YQKcrST H3K56 CYQKSTELL 14 CYQKcrSTELL H3K56LLPKKTESHHKAK 15 LLPKKcrTESHHKAK H2AK119 CLLPKKTESHHKAKG 16CLLPKKcrTESHHKAKG H2AK119 APAPKKGS 17 APAPKcrKGS H2BK11 APAPKKGSC 18APAPKcrKGSC H2BK11 APAPKKGS 17 APAPKKcrGS H2BK12 CAPAPKKGS 19CAPAPKKcrGS H2BK12 GSKKA 20 GSKKcrA H2BK16 GSKKAVTC 21 GSKKcrAVTC H2BK10TKAQKKDG 22 TKcrAQKKDG H2BK20 AVTKAQKKDGC 23 AVTKcrAQKKDGC H2BK20ARTKQTAR 24 ARTKcrQTAR H3K4 ARTKQTARC 25 ARTKcrQTARC H3K4 APRKQLA 26APRKcrQLA H3K18 APRKQLATC 27 APRKcrQLATC H3K18 QLATKAARK 28 QLATKcrAARKH3K23 QLATKAARKC 29 QLATKcrAARKC H3K23 AAPKSAP 30 AARKcrSAP H3K27AARKSAPATGGC 31 AARKcrSAPATGGC H3K27 CRLLRKGNYAER 32 CRLLRKcrGNYAERH2AK36 CLLPKKTESHHKAKG 16 CLLPKcrKTESHHKAKG H2AK118 CLLPKKTESHHKAKG 16CLLPKKTESHHKcrAKG H2AK125 CAVTKAQKKDG 33 CAVTKAQKcrKDG H2BK23CARTKQTARKSTG 34 CARTKQTARKcrSTG H3K9 CSGRGKGG 35 CSGRGKcrGG H4K5CGLGKGGAKRHR 36 CGLGKGGAKcrRHR H4K16 CGTPRKASGP 37 CGTPRKcrASGP H1K34LKKALAAAGYC 38 LKKcrALAAAGYC H1K64 CKLGLKSLVSK 39 CKLGLKcrSLVSK H1K85CKSLVSKGTL 40 CKSLVSKcrGTL H1K90 CVQTKGTGA 41 CVQTKcrGTGA H1K97CTPKKAKKPA 42 CTPKKAKcrKPA H1K159 VTKKVAKSPKC 43 VTKcrKVAKSPKC H1K168

TABLE 2  Modifications on Adjacent Residues of Histone Kcr PeptidesCrotonylation Peptide Sequence Crotonylated Peptide SitePeptide sequence PTM site CRLLRKGNYAER CRLLRKcrGNYAER H2AK366CRLLRKGNYoxAER H2AY39 CLLPKKTESHHKAKG CLLPKcrKTESHHKAKG H2AK118CLLPKKphoTESHHKAKG H2AT120 CLLPKKTESHHKAKG CLLPKKcrTESHHKAKG H2AK119CLLPKKTESHHKAKG CLLPKKTESHHKcrAKG H2AK125 APAPKKGSC APAPKcrKGSC H2BK11APAPKKGSphoC H2BS14 CAPAPKKGS CAPAPKKcrGS H2BK12 GSKKAVTC GSKKcrAVTCH2BK16 GSK(me, ac)KAVTC H2BK15 AVTKAQKKDGC AVTKcrAQKKDGC H2BK20AVTKAQKKacDGC H2BK24 CAVTKAQKKDG CAVTKAQKcrKDG H2BK23 CRSRKESYCRSRKcrESY H2BK34 CRSphoRKcrESphoYox H2BK32, 36, 37 ARTKQTARCARTKcrQTARC H3K4 ARTphoKQTARmeC H3K3, 8 CARTKQTARKSTG CARTKQTARKcrSTGH3K9 CARTKQTARKSphoTphoG H3K10, 11 APRKQLATC APRKcrQLATC H3K18APRmeKQLATC E3K17 QLATKAARKC QLATKcrAARKC H3K23 QLATKAARmeKC H3K26AARKSAPATGGC AARKcrSAPATGGC H3K27 AARKSphoAPATGGC H3K28 CYQKSTELLCYQKcrSTELL H3K56 CSGRGKGG CSGRGKcrGG HK4K5 CSphoGRmeGKGG H4K1, 3GGKGLGKC GGKcrGLGKC H4K8 GGKGLGK(succ, pro, H4K12 bu, ac, me)CCGLGKGGAKRHR CGLGKGGAKcrRHR H4K16 CRLLRKGNYAER CRLLRKcrGNYAER H2AK36CRLLRKGNYoxAER H2AY39 CLLPKKTESHHKAKG CLLPKcrKTESHHKAKG H2AK118CLLPKKTphoESHHKAKG H2AT120 CLLPKKTESHHKAKG CLLPKKcrTESHHKAKG H2AK119CLLPKKTESHHKAKG CLLPKKTESHHKcrAKG H2AK125 APAPKKGSC APAPKcrKGSC H2BK11APAPKKGSphoC H2BS14 CAPAPKKGS CAPAPKKcrGS H2BK12 GSKKAVTC GSKKcrAVTCH2BK16 GSK(me, ac)KAVTC H2BK15 AVTKAQKKDGC AVTKcrAQKKDGC H2BK20AVTKAQKKacDGC H2BK24 CAVTKAQKKDG CAVTKAQKcrKDG H2BK23 CRSRKESYCRSRKcrESY H2BK34 CRSphoRKcrESphoYox H2BK32, 36, 37 ARTKQTARCARTKcrQTARC H3K4 ARTphoKQTARmeC H3K3, 8 CARTKQTARKSTG CARTKcrQTARKSTGH3K9 CARTKQTARKSphoTphoG H3K10, 11 APRKQLATC APRKcrQLATC H3K18APRmeKQLATC H3K17 QLATKAARKC QLATKcrAARKC H3K23 QLATKAARmeKC H3K26AARKSAPATGGC AARKcrSAPATGGC H3K27 AARKSphoAPATGGC H3K28 CYQKSTELLCYQKcrSTELL H3K56 CSGRGKGG CSGRGKcrGG H4K5 CSphoGRmeGKGG H4K1, 3GGKGLGKC GGKcrGLGKC H4K8 GGKGLGKC(succ, pro, H4K12 bu, ac, me)CGLGKGGAKRHR CGLGKGGAKcrRHR H4K16 CGTPRKASGP CGTPRKcrASGP H1K34LKKALAAAGYC LKKcrALAAAGYC H1K64 LK(me, ac)KALAAAGY H1K63, 71 (ox)CCKLGKSLVSK CKLGKcrSLVSK H1K85 CK(fo)LGKSLVSK H1K81, 90 (me, ac)CKSLVSKGTL CKSLVSKcrGTL H1K90 CVQTKGTGA CVQTKcrGTGA H1K97 CTPKKAKKPACTPKKAKcrKPA H1K159 VTKKVAKSPKC VTKcrKVAKSPKC H1K168 Ox: oxidation; pho:phosphorylation; me: methylation; succ: succinylation; pro:propionylation; bu: butyrylation; ac: acety.

What is claimed:
 1. An isolated peptide comprising a crotonylation site.2. The isolated peptide of claim 1, wherein the peptide is derived froma histone protein or a fragment thereof.
 3. The isolated peptide ofclaim 1, wherein the peptide comprises a sequence selected from SEQ IDNOs: 11-43.
 4. The isolated peptide of claim 1, wherein the peptide iscrotonylated at a lysine site.
 5. The isolated peptide of claim 4,wherein the crotonylated peptide is selected from the group consistingof QLATKcrAA, CQLATKcrAA, YQKcrST, CYQKcrSTELL, LLPKKcrTESHHKAK,CLLPKKcrTESHHKAKG, APAPKcrKGS, APAPKcrKGSC, APAPKKcrGS, CAPAPKKcrGS,GSKKcrA, GSKKcrAVTC, TKcrAQKKDG, AVTKcrAQKKDGC, ARTKcrQTAR, ARTKcrQTARC,APRKcrQLA, APRKcrQLATC, QLATKcrAARK, QLATKcrAARKC, AARKcrSAP,AARKcrSAPATGGC, CRLLRKcrGNYAER, CLLPKcrKTESHHKAKG, CLLPKKTESHHKcrAKG,CAVTKAQKcrKDG, CARTKQTARKcrSTG, CSGRGKcrGG and CGLGKGGAKcrRHR.
 6. Theisolated peptide of claim 1, wherein the crotonylation site is selectedfrom the group consisting of human H1.2K33, H1.2K63, H1.2K84, H1.2K89,H1.2K96, H1.2K158, H1.2K167, H2AK36, H2AK118, H2AK119, H2AK125, H2BK5,H2BK11, H2BK12, H2BK5, H2BK16, H2BK20, H2BK23, H2BK34, H3K4, H3K9,H3K18, H3K23, H3K27, H3K56, H4K5, H4K8, H4K12 and H4K16.
 7. The isolatedpeptide of claim 1, wherein the crotonylation site is selected from thegroup consisting of human H2AK119, H2BK11, H2BK12, H2BK16, H2BK20,H2BK34, H3K4, H3K18, H3K23, and H3K27.
 8. A method for producing aKcr-specific affinity reagent that binds specifically to a protein or afragment thereof comprising a crotonylation site, wherein theKcr-specific affinity reagent is an antibody, comprising immunizing ahost with the peptide of claim
 1. 9. The method of claim 8, wherein thepeptide comprises a sequence selected from SEQ ID NOs: 11-43.
 10. Amethod for producing a Kcr-specific affinity reagent that bindsspecifically to a protein or a fragment thereof comprising acrotonylation site, wherein the Kcr-specific affinity reagent is apolypeptide, comprising screening a synthetic peptide library using thepeptide of claim
 1. 11. The method of claim 10, wherein the syntheticpeptide library is a phage display library or a yeast display library.12. The method of claim 11, wherein the peptide comprises a sequenceselected from SEQ ID NOs: 11-43.
 13. An isolated Kcr-specific affinityreagent capable of binding specifically to the peptide of claim
 1. 14.The isolated Kcr-specific affinity reagent of claim 13, wherein thepeptide comprises a sequence selected from SEQ ID NOs: 11-43.
 15. Theisolated Kcr-specific affinity reagent of claim 13, wherein the bindingof the Kcr-specific affinity reagent is dependent on the crotonylationsite but not its surrounding peptide sequence.
 16. The isolatedKcr-specific affinity reagent of claim 13, wherein the binding of theKcr-specific affinity reagent is dependent on the crotonylation site andits surrounding peptide sequence.
 17. The isolated Kcr-specific affinityreagent of claim 13, wherein the Kcr-specific affinity reagent bindsspecifically to the peptide when crotonylated at the crotonylation site,and wherein the Kcr-specific affinity reagent does not bind to thepeptide when not crotonylated at the crotonylation site.
 18. Theisolated Kcr-specific affinity reagent of claim 13, wherein thecrotonylation site is selected from the group consisting of humanH1.2K33, H1.2K63, H1.2K84, H1.2K89, H1.2K96, H1.2K158, H1.2K167, H2AK36,H2AK118, H2AK119, H2AK125, H2BK5, H2BK 11, H2BK12, H2BK15, H2BK16,H2BK20, H2BK23, H2BK34, H3K4, H3K9, H3K18, H3K23, H3K27, H3K56, H4K5,H4K8, H4K12 and H4K16.
 19. A method for detecting protein crotonylationin a sample, comprising: (a) contacting the sample with the isolatedKcr-specific affinity reagent of claim 13 to form a binding complex,whereby the Kcr-specific affinity reagent binds specifically to aprotein or a fragment thereof comprising a crotonylation site when theprotein or a fragment thereof is crotonylated at the crotonylation site,and (b) detecting the binding complex, wherein the presence of thebinding complex indicates protein crotonylation in the sample.
 20. A kitfor detecting protein crotonylation in a sample, comprising the isolatedKcr-specific affinity reagent of claim 13, wherein the Kcr-specificaffinity reagent binds specifically to a protein or a fragment thereofcomprising a crotonylation site when the protein or a fragment thereofis crotonylated at the crotonylation site.